microbial degradation of quaternary ammonium alcohols

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Doctoral Thesis

Microbial degradation of quaternary ammonium alcoholshydrolysis products of esterquat surfactants used as fabricsofteners

Author(s): Käch, Andres

Publication Date: 2002

Permanent Link: https://doi.org/10.3929/ethz-a-004423464

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DISS. ETH NO. 14757

Microbial degradation of quaternary ammonium alcohols

hydrolysis products of esterquat surfactants used as fabric softeners

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

for the degree of

Doctor ofNatural Sciences

presented by

ANDRESKAECH

Dip!. Natw. ETH

born 10.03.1969

Citizen ofEmmen, Lucerne

accepted on the recommendation of

Prof. Dr. A. J. B. Zehnder, examiner

Dr. N. Rehman, co-examiner

PD Dr. T. Egli, co-examiner

Zurich, 2002

.(1' ite Leer /I

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Dank

Allen voran mochte ich PD Dr. Thomas Egli fUr die Betreuung und Untersttitzung wahrend

meiner Dissertation sowie fUr seinen immerwahrenden Optimismus danken.

Dank gebtihrt auch:

Prof. Alexander Zehnder fUr die Ubemahme des Referates und wertvolle Diskussionen.

Steve G. Hales fUr die Initiation dieser Arbeit, Naheed Rehman fUr die FortfUhrung der

Betreuung seitens Unilever und die Ubemahme des Korreferates sowie Unilever (SEAC

Applied Science & Technology, Unilever Colworth, UK) fUr die Finanzierung dieser Arbeit.

Martina Hofer, die mit ihrer Diplomarbeit einen wesentlichen Beitrag an diese Arbeit geleistet

und mich fUr meine letzte Laborzeit nochmals so richtig motiviert hat.

Nathalie Vallotton fUr ihren grossen Beitrag an die Charakterisierung der isolierten Bakterien

wahrend ihres Praktikums.

Henri Lambert fUr die wertvolle Arbeit wahrend seiner Diplomarbeit.

Dem Laborantenteam, namentlich Thomi, Hansueli, Christoph, Teresa, Karin, Andy und

Bettina fUr ihre Hilfsbereitschaft, technische Beratung und Gesellschaft.

Der Gruppe Egli fUr das angenehme Arbeitsklima und hilfreiche Diskussionen und

insbesondere allen MitbastlerInnen im F 51, welche wesentlich zur Verarbeitung meiner

Hohen und Tiefen beigetragen haben, speziell Lukas, meinem langjahrigen Labomachbar.

Dani Rentsch fUr die Messung und Interpretation unzahliger NMR Spektren und fUr wertvolle

sowie kritische Diskussionen.

Wemer Angst fUr die Beratung in organisch chemischen Fragen.

Dem ganzen Prozess MIX fUr die gute Atmosphare.

Den Bibliothekarinnen fUr ihre stete Hilfsbereitschaft und prompten Lieferungen der

manchmal nicht leicht zu bekommenden Literatur.

Meinen Eltem, welche mich bisher in allem, was ich untemommen habe, jederzeit untersttitzt

haben.

"'- ._------,

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Publications

The following part of this thesis has been published:

Kaech, A. & Egli, T. (2001). Isolation and characterization of a Pseudomonas putida strain

able to grow with trimethyl-l,2-dihydroxy-propyl-ammonium as sole source of carbon,

energy and nitrogen. Syst Appl Microbiol24, 252-261. (Chapter 2)

The following publications are in preparation:

Kaech, A., Vallotton, N. & Egli, T. Isolation and characterisation of microorganisms able to

grow with quaternary ammonium alcohols as sole source of carbon, energy and nitrogen.

(Chapter 3)

Kaech, A., Hofer, M., Rentsch, D. & Egli, T. Microbial oxidation of methyl-triethanol

ammonium. (Chapter 4)

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Table of contents

Table of contents

Abbreviations 9

Summary 11

Zusammenfassung 13

1. General introduction 17

2. Isolation and characterisation of a Pseudomonas putida strain able to grow

with 2,3-dihydroxypropyl-trimethyl-ammonium as sole source of carbon,

energy and nitrogen 23

3. Isolation and characterisation of bacteria able to grow with quaternary

ammonium alcohols as sole source of carbon, energy and nitrogen 43

4. Microbial oxidation of methyl-triethanol-ammonium 65

5. Microbial degradation of 2,3-dihydroxypropyl-trimethyl-ammonium 89

6. Concluding remarks 111

References 117

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Abbreviations

BSA

BTF

CE

CFE

CHD

D

DM

DOC

DON

dpm

Glycidol

INT

KEGG

MM

OD

OECD

PB

PF

PHA

PMS

QAA

SF

SM

TM

TMA

TSA

TSB

TSP

Bovine serum albumin

Benzotrifluorid

Crude extract

Cell-free extract

Choline dehydrogenase

Dilution rate

Dimethyl-diethanol-ammoniurn

Dissolved organic carbon

Dissolved organic nitrogen

Decays per minute

(±)-Oxiran-2-methanol

Iodonitrotetrazolium chloride

Kyoto Encyclopedia of Genes and Genomes

Methyl-triethanol-ammonium

Optical density

Organization for Economic Cooperation and Development

Phosphate buffer (50 mM)

Particulate fraction of CFE

Polyhydroxyalkanoate

Phenazine methosulfate

Quaternary ammonium alcohol

Soluble fraction of CFE

Synthetic medium

(±)-2,3-Dihydroxypropyl-trimethyl-ammonium

Trimethylamine

Tryptic soy agar

Tryptic soy broth

Sodium 3-trimethylsilyltetradeutero-propionate

9

Abbreviations

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1..-- . __..__

Summary

Summary

The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium

(TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are the

mainly used head groups in esterquat surfactants, which are widely applied as softeners in

fabric care. Four bacterial strains able to grow with these QAAs as the sole source of carbon,

energy and nitrogen were isolated. One strain was isolated with each, TM and MM, referred

to as strain TM 1 and MM 1, respectively. Two strains were isolated with DM, designated as

strain DM 1 and DM 2. Phylogenetic identification was performed and the morphology of

cells and colonies, the nutritional and biochemical properties as well as the growth

characteristics were investigated. Phylogenetic analysis (16S-rDNA) revealed for strain TM 1

identities to Pseudomonas putida DSM 291T of 99.9 % (closest relationship). For the strains

DM 1 and DM 2 the analysis provided closest relationship to Zoogloea ramigera Itzigsohn

1868AL of 97 and 98 % identities, respectively. Closest related strain for isolate MM 1 was

found to be Rhodobacter sphaeroides with identities of 94 % only. However, anaerobic

growth in the light and the formation of pigments characteristic for all representatives of the

genus Rhodobacter was not observed for strain MM 1. Therefore, strain MM 1 must be

considered to be a member of a new genus.

Out of the four investigated strains, only strain DM 2 isolated with DM was able to grow with

another QAA (TM) than that used for its isolation. No consortia of microorganisms were

required for complete degradation of the three QAAs. All strains were able to grow with the

naturally related compound choline, but none of the choline degrading bacterial reference

strains tested was able to grow with any of the QAAs. Therefore, the ability to degrade

choline does not go along with the competence to catabolise the three QAAs. The isolated

strains belonged to different genera and hence, the degradation of QAAs is not a trait of a

single microbial genus.

The primary catabolism of the QAAs was investigated and the metabolites were identified by

NMR spectroscopy. The initial enzymatic attack on MM in isolate MM 1 was mediated by a

membrane-associated, constitutively expressed oxidoreductase, oxidising ethanol groups first

to the corresponding aldehydes and then to the carboxylic acids. However, as soon as one

ethanol group of MM was oxidised to the aldehyde, a cyclisation occurred intramolecularly

with a second ethanol group forming a cyclic hemiacetal. No further oxidation of the cyclic

11

Summary

hemiacetal was observed. Only the remaining third ethanol group of MM was oxidised to the

aldehyde and to the carboxylic acid. The cyclic hemiacetal products with the third ethanol

group oxidised twice to the carboxylic acid appeared to be dead-end metabolites, since in

batch cultures of strain MM I considerable amounts of this compound were released to the

medium and remained untouched. Hence, these metabolites may accumulate in the

environment. The oxidation of the ethanol groups of DM and choline proceeded under the

same assay conditions as for the oxidation of MM, providing one cyclic hemiacetal product

from DM and betainealdehyde and betaine from choline. This suggests that the same enzyme

was responsible for the oxidation of MM, DM and choline. It might be a choline

oxidoreductase with extended substrate specificity.

In contrast, the initial attack on TM in isolate TM 1 was catalised by an inducible, membrane

associated lyase removing trimethylamine from the molecule. In the cell-free extracts of this

strain, enzymatic transformation of neither DM, MM, betaine nor carnitine was observed.

Only choline was transformed, however, it did not undergo a C-N fission as detected for TM,

but was oxidised to betainealdehyde and betaine, as found in the MM-growing strain MM 1.

Therefore, in strain TM 1 the mechanisms for the initial attack on TM and choline,

respectively, are different. When grown with TM, the fission of the C-N bond of TM and the

release of trimethylamine were also found in the cell-free extracts of the isolate DM 2 able to

grow with DM and TM, although this strain belongs to a different genus.

In the cellular fractions of the DM-growing strains DM 1 and DM 2 no DM-consuming

activity was found and therefore, no enzymatic investigation of the catabolism of DM was

possible.

Based on these findings, no general strategy in microorganisms can be proposed for the

degradation of QAAs despite the similarity of these compounds in their chemical structure.

Also, no general relationship seems to exist between the degradation of the QAAs and the

naturally related compound choline. The ability to degrade the QAAs appears to be a specific

capability of specialised microorganisms, and individual degradation mechanisms seem to be

followed for each of the three QAAs.

12

Zusammenfassung

Zusammenfassung

Die quatemaren Ammoniumalkohole (QAAs) 2,3-Dihydroxypropyl-trimethyl-ammonium

(TM), Dimethyl-diethanol-ammonium (DM) und Methyl-triethanol-ammonium (MM) sind

die meist verwendeten Kopfgruppen in Esterquat-Tensiden, welche als Weichmacher in

grossen Mengen in der Textilreinigung eingesetzt werden und weit verbreitet sind. Vier

bakterielle Stamme, welche mit den entsprechenden QAAs als alleinige Kohlenstoff-,

Energie- und Stickstoffquelle wachsen konnen, wurden isoliert. Je ein Stamm wurde mit TM

und MM isoliert, bezeichnet als Stamm TM 1 beziehungsweise MM 1. Mit DM wurden zwei

Stamme isoliert, Stamm DM 1 und DM 2. Die Isolate wurden phylogenetisch identifiziert,

und die Morphologie der Zellen und Kolonien, das Niihrstoffspektrum, diverse biochemische

Eigenschaften sowie die Wachstums-Charakteristik wurden eingehend untersucht. Die

phylogenetische Analyse (16S-rDNA) ergab fur den Stamm TM 1 eine Obereinstimmung von

99.9 % mit Pseudomonas putida DSM 291 T (nachste Verwandtschaft). Fiir die Stamme DM 1

und DM 2 lieferte die Analyse eine nachste Verwandtschaft zu Zoogloea ramigera Itzigsohn

1868AL mit einer Obereinstimmung von 97 beziehungsweise 98 %. Der nachst verwandte

Stamm von Isolat MM 1 war Rhodobacter sphaeroides mit einer Obereinstimmung von nur

94 %. Da weder anaerobes Wachstum mit Licht noch die Bildung von Pigmenten fur Stamm

MM 1 beobachtet wurde, beides Eigenschaften aller Vertreter der Gattung Rhodobacter, ist

Isolat MM 1 hochst wahrscheinlich ein Vertreter einer neuen Gattung. Von den vier isolierten

Bakterienstammen war nur Isolat DM 2, isoliert mit DM, dazu befahigt, mit einem der

anderen QAAs (TM) zu wachsen. Es waren keine Konsortien von verschiedenen Bakterien

fur den Abbau der drei QAAs notwendig. Alle isolierten Bakterien konnten mit der

natiirlichen, strukturell verwandten Substanz Cholin wachsen. Jedoch konnte keiner der

getesteten cholinabbauenden bakteriellen Referenzorganismen mit einem der QAAs wachsen.

Daher impliziert die Fahigkeit Cholin abzubauen nicht notwendigerweise die Fahigkeit, auch

die QAAs zu verwerten. Da die isolierten Sllimme zu verschiedenen Gattungen gehoren, ist

der Abbau der QAAs nicht die Vorherrschaft einer einzelnen bakteriellen Gattung.

Der anfangliche Abbaustoffwechsel der QAAs wurde untersucht und die

Umwandlungsprodukte wurden mit Hilfe der NMR-Spektroskopie identifiziert. Der initiale

enzymatische Abbauschritt von MM in Isolat MM 1 wurde durch eine membrangebundene,

konstitutiv exprimierte Oxidoreduktase katalysiert, welche Ethanolgruppen zuerst zum

13

Zusammenfassung

entsprechenden Aldehyd und dann zur Carboxysaure oxidiert. Sobald jedoch eine

Ethanolgruppe von MM zum Aldehyd oxidiert war, fand eine intramolekulare Zyklisierung

mit einer zweiten Ethanolgruppe statt, welche zu einem zyklischen Hemiacetal fUhrte. Eine

weitere Oxidation des zyklischen Hemiacetals wurde nicht beobachtet. Nur die

tibrigbleibende dritte Ethanolgruppe von MM wurde zum Aldehyd und zur Saure oxidiert.

Die zyklischen Hemiacetal-Produkte, deren dritte Ethanolgruppe jeweils zur Carboxysaure

oxidiert war, scheinen Sackgasse-Abbauprodukte zu sein, da in Batchkulturen des Stammes

MM 1 erhebliche Mengen dieser Zwischenprodukte ausgeschieden und nicht weiterverwertet

wurden. Diese Zwischenprodukte konnten sich daher hypothetisch in der Umwelt anreichem.

Die Oxidation der Ethanolgruppen von DM und Cholin erfolgte unter den gleichen

experimentellen Bedingungen wie die Oxidation von MM, wobei hauptsachlich ein

zyklisches Hemiacetal-Produkt aus DM und Betainaldehyd und Betain aus Choline gebildet

wurde. Daher war das aktive Enzym vermutlich dasselbe fUr MM, DM und Cholin. Es konnte

sich urn eine Cholin Oxidoreduktase mit erweitertem Substratspektrum handeln.

Im Gegensatz dazu beruhte der initiale Abbauschritt von TM in Stamm TM 1 auf einem

anderen Mechanismus. Es wurde eine induzierbare, membrangebundene Lyaseaktivitat

gefunden, welche Trimethylamin von TM abspaltet. In den zellfreien Extrakten dieses

Stammes wurden weder DM, MM, Betain noch Camitin umgewandelt. Nur Cholin wurde

abgebaut, jedoch nicht durch eine Spaltung der C-N Bindung wie bei TM, sondem durch eine

Oxidation zu Betainaldehyd und Betain, wie dies auch im MM-wachsenden Stamm MM 1

beobachtet wurde. Damit sind die Mechanismen des initialen Abbauschrittes von TM

beziehungsweise Cholin in Stamm TM 1 ganzlich unterschiedlich. Die Spaltung der C-N

Bindung von TM und die Freisetzung von Trimethylamin wurde auch im zellfreien Extrakt

des mit TM gewachsenen Stammes DM 2 gefunden, welcher mit DM und TM wachsen kann,

obwohl dieser Stamm zu einer anderen Gattung gehort.

In den Zellaufschltissen der mit DM wachsenden Stamme DM 1 und DM 2 wurde keine

Enzymaktivitat fUr DM gefunden und deshalb konnten keine enzymatischen Untersuchen

beztiglich des Abbaustoffwechsels von DM durchgeftihrt werden.

Aufgrund dieser Resultate kann trotz der ahnlichen chemischen Struktur dieser Substanzen

keine generelle Strategie in Mikroorganismen fUr den Abbau der QAAs vorgeschlagen

werden. Zudem scheint keine allgemeine Beziehung zwischen dem Abbau der QAAs und der

14

Zusammenfassung

nattirlich verwandten Substanz Cholin zu bestehen. Die Fahigkeit, QAAs abzubauen, ist

offenbar eine Fahigkeit von spezialisierten Mikroorganismen, und die gewahlten

Abbaumechanismen scheinen von der spezifischen Struktur der QAAs abhangig zu sein.

15

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General introduction

1. General introduction

In the 1950s and 1960s, a new group of surfactants was successfully introduced in the

detergent and cleaning agent sector. These were called softeners because they eliminated the

harsh feel produced by modern laundry processes. Softeners, however, do not only soften

tissues. They have a number of other useful properties such as preventing accumulation of

electrostatic charge, improving the suppleness of the fibers, making ironing easier, shorten

drying time, and they impart a pleasant fragrance to laundry. Because of the many

advantageous properties and effects, they very soon became popular and, hence, they present

now a very important group of products (Puchta et aI., 1993).

Not many different chemicals are used as softeners. For more than three decades (up to 1990)

almost exclusively one raw material was applied. This material was called DTDMAC

ditallow-dimethyl-ammonium chloride. It is a quaternary ammonium compound with two

methyl groups and two alkyl groups derived from tallow connected to the nitrogen atom

(Figure 1. la). Commercial DTDMAC typically comprised C18 (65 %), C16 (30 %) and C14

(5 %) alkyl chains (Sullivan, 1982). In the 1990s DTDMAC was given an environmentally

hazardous classification as a result from laboratory studies suggesting that the Predicted

Environmental Concentration (PEC) of DTDMAC would exceed its No-Effect Concentration

(NEC), particularly in surface waters charged heavily with treated wastewater. Although a

detailed study published later by the European Center for Ecotoxicology and Toxicology of

Chemicals (ECETOC) came to the conclusion that the use of DTDMAC is safe and its

environmental concentrations do not pose a risk to aquatic and terrestrial ecosystems, the

industry at that time reacted rapidly and a new group of softeners with improved ecological

properties was introduced in several countries (Berenbold, 1990; Krueger et aI., 1998; Puchta

et al., 1993). The difference to the DTDMAC based softeners consisted in the insertion of

esters groups into the alkyl chains, and due to this chemical structure they were named

esterquat surfactants. Three similar esterquat surfactants were developed and are still today

the mainly used softeners (Figure 1.1b). The annual production exceeded in the 1990s

probably 100'000 tons worldwide, with basically over 99 % of the material being used in

fabric care (Krueger et aI., 1998). These esterquat softeners have proven to fulfill all the

properties required for good washing. Based on standardised test procedures, they were

expected to be readily and ultimately biodegradable and to cause no risks for human health

17

Chapter 1

(a) DTDMAC*

(b) Esterquat surfactants

oOAR

,,( I/~OyR

oTM-esterquat

tdrOIYSiS

~ ........ fatty acid

DM-esterquat

tdrOIYSiS

~ ........ fattYacid

MM-esterquat

tdrOIYSiS

~ ........ fatty acid

(c) Quaternary ammonium alcohols (QAAs)

/ OH

"N~OH/

TM

HO /"--.../OH~N+

/~OH

MM

(d) Choline, structurally related compound of natural origin

Rand *: Fatty acid carbon chain derived from tallow, mainly C16 and C1S.

Figure 1.1. Structure of Ca) DTDMAC, Cb) the esterquat surfactants used as fabric softeners, Cc) their

hydrolysis products, Cd) and the structurally related compound choline.

(Giolando et al., 1995; Krueger et al., 1998; Matthijs et al., 1995; Puchta et al., 1993; Waters

et aI., 1991; Waters et al., 2000).

The ester bonds, characteristic for these new softeners, serve as potential breaking points of

the molecule and, therefore, contribute to the good environmental behaviour of these

compounds (Figure 1.1b) (Puchta et al., 1993). They hydrolise rapidly, abiotically and/or

biocatalysed, when reaching surface water or sewage treatment plants. The resulting products

are the fatty acids and the corresponding quaternary ammonium alcohols (QAAs)

2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and

methyl-triethanol-ammonium (MM) as displayed in Figure 1.1c. The fatty acids are common

compounds in the environment and hence are degraded readily and completely by many

different microorganisms by ~-oxidation. The QAAs were investigated as well by applying

18


the same standardised test procedures as for the parent compounds and were also expected to

be readily and ultimately biodegradable in the environment and to have no harmful effects on

human health (Giolando et al., 1995; Hellberg et al., 2000; Krueger et al., 1998; Matthijs et

al., 1995; Puchta et al., 1993; Simms et al., 1992; Waters et al., 2000). However, it is still

possible that one of the QAAs might accumulate in the environment due to high amounts used

of the esterquat surfactants.

Surprisingly, the three structurally similar QAAs showed very different degradation patterns

and degradation rates in OECD die-away tests (OECD, 1981) mimicking complex

environmental systems, although all three QAAs were assessed as readily and ultimately

biodegradable (Hales, 1998). Considering the similarity of the three QAAs and their similarity

to the naturally related compound choline (Figure 1.1d), one could suggest that all of the three

QAAs should degrade in a similar way and with a rate comparable to that of choline.

However, this was not the case. The question arised as to why the three QAAs behave

differently concerning their biodegradation and what mechanisms might be responsible for

their degradation. Was it due to the limited distribution and occurrence of QAA degrading

microbes or to their QAA degrading properties? Furthermore, since choline is a widespread

compound among microorganisms as well as in higher organisms (Kortstee, 1970) the

question came up whether the mechanisms of QAA breakdown are related to those involved

in the degradation of choline.

Up to now, no microorganisms degrading the three QAAs TM, DM and MM have been

isolated and consequently the catabolic pathways are not elucidated yet. Knowledge of these

mechanisms is very important considering the high amounts of these compounds used and the

increasing market of a whole group of similar compounds. Moreover, a better understanding

could affect the selection of more favorable and the design of new similar compounds, which

are not only used as raw material for softeners but also for cosmetics, drugs and other

chemicals used in biological applications (Krueger et al., 1998; Vievsky, 1997).

Several studies report on the microbial degradation of choline. The general degradation

pathway (Figure 1.2a) present in many and widely different microbial strains proceeds by

successive oxidation to betainealdehyde and betaine, followed by progressive demethylation

to dimethylglycine, sarcosine and finally the amino acid glycine (Kortstee, 1970; Shieh, 1964;

19

Chapter 1

KEGG Kyoto Encyclopedia of Genes and Genomes, www.genome.ad.jp). Several bacterial

enzymes mediating initial choline oxidation have been described in the literature (Bater &

Venables, 1977; Haubrich & Gerber, 1981; Ikuta et al., 1977; Kiene, 1998; Nagasawa et aI.,

1975; Nagasawa et aI., 1976; Ohta-Fukuyama et aI., 1980; Rosenstein et al., 1999; Yamada et

aI., 1979). Additionally to initial choline oxidation, a fission of the C-N bond, producing

trimethylamine, was observed in Proteus vulgaris (Seim et aI., 1982b) and Shigella

alkalescens (Wood & Keeping, 1944). In both cases, trimethylamine was released into the

culture medium.

In contrast, only a limited number of studies on the isolation of bacteria able to degrade

human-made quaternary ammonium compounds have been reported so far (Dean-Raymond &

Alexander, 1977; Nishihara et aI., 2000; Van Ginkel et aI., 1992). Microorganisms were

isolated from activated sewage sludge or soil using the quaternary ammonium surfactants

decyl-trimethyl-ammonium, hexadecyl-trimethyl-ammonium and didecyl-dimethyl

ammonium as sole source of carbon. However, the breakdown of these compounds by pure

cultures was always incomplete and required a consortium of at least two different

microorganisms for complete degradation. Based on these studies and other investigations on

the biodegradation of a series of anionic, non-ionic, amphoteric and cationic surfactants Van

Ginkel (1996) concluded that complete degradation of most surfactants has to be achieved by

consortia of microorganisms. This author suggested that only a few surfactants, i. e. alkane

sulphonates, alkyl sulphates and alkylamines are completely degraded by pure microbial

cultures. Three possible degradation mechanisms of alkyl-trimethyl-ammonium compounds

have been postulated by Van Ginkel (1995) (Figure 1.2b):

(1) Oxidation at the free end of the alkyl chain followed by stepwise degradation via

~-oxidation (fatty acid metabolism) resulting in betaine, which is oxidatively

demethylated and finally results in the amino acid glycine (Figure 1.2a).

(2) Progressive demethylation of the nitrogen centre, then the splitting off of the nitrogen

from the alkyl chain in the form of ammonia. The alkyl chain again is degraded by

~-oxidation.

(3) Oxidative cleavage of the N-Calkyl bond providing trimethylamine and the aldehyde of the

alkyl chain. Whereas the alkyl chain again undergoes ~-oxidation, trimethylamine is

degraded by methylotrophic organisms.

20


(a)

/ "U/ OH

"N+ N+ II "N~/~OH • / 0 •

III III / 0

Choline Betainealdehyde Betaine

IvlOH VI I OH V I OH

H2N~O .. HN~ .. /N~OVII 0

Glycine Sarcosine Dimethylglycine

(b)

o

H~~Betaine

Figure 1.2. (a) General schematic degradation pathway of choline with responsible enzymes, adapted

from the Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp): I) Choline dehydrogenase

(EC 1.1.99.1), II) betainealdehyde dehydrogenase (EC 1.2.1.8), Ill) choline oxidase (EC 1.1.3.17), IV)

betaine homocysteine S-methyltransferase (EC 2.1.1.5), V) dimethylglycine dehydrogenase

(EC 1.5.99.2), VI) sarcosine dehydrogenase (EC 1.5.99.1), VII) sarcosine oxidase (EC 1.5.3.1).

(b) Schematic degradation mechanisms of alkyl-trimethyl-ammonium compounds as proposed by Van

Ginkel (1995); numbers correspond to the explanations in the text.

21

Chapter 1

Van Ginkel (1996) proposed that the initial fission of the N-Calkyl bond represents a general

strategy of microorganisms to gain access to the alkyl chains of quaternary ammonium

compounds.

The primary goal of this thesis was the elucidation of the strategies of microorganisms to

degrade the quaternary ammonium alcohols TM, DM and MM. To approach this goal, the

isolation of competent microorganisms degrading the QAAs is an indispensable requirement,

since these bacteria are the basis for further investigations at the enzyme level. With respect to

the aim set the following questions were considered to be of primary importance:

• Can pure microbial cultures or defined consortia be enriched and isolated that are able to

grow with the three QAAs TM, DM and MM as sole sources of carbon, energy and

nitrogen and are capable to degrade them to completion?

• Are the strains isolated on the different QAAs totally different, similar, or even the same?

• How specific is the degradation of the different QAAs. Are the different isolated strains

able to degrade the two other QAAs, not used for their isolation, too?

• Are the strains able to grow with the naturally related compound choline?

• What kinds of enzymes are responsible for the first step in the catabolism of the individual

QAAs?

• Are the enzymes of the different microorganisms, attacking the different QAAs, the same

or different?

• How specific are the enzymes with respect to structurally similar compounds?

• Are the enzymes similar or identical to those responsible for the degradation of choline?

22

Pseudomonas putida TM 1

2. Isolation and characterisation of a Pseudomonas putida strain able to

grow with 2,3-dihydroxypropyl-trimethyl-ammonium as sole source of

carbon, energy and nitrogen

ABSTRACT

2,3-dihydroxypropyl-trimethyl-ammonium (TM) originates from the hydrolysis of the parent

esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures

mimicking complex biological systems, TM is supposed to degrade completely when

reaching the environment. However, no organisms able to degrade TM were isolated nor has

the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to

grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum

specific growth rate of 0.4 h- l when growing with TM as the sole source of carbon, energy

and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as

ammonium into the growth medium. A high percentage of the carbon in TM (68 % in

continuous culture and 60 % in batch culture) was combusted to C02 resulting in a low yield

of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry

weight per mg carbon in batch cultures. Choline, a natural structurally related compound,

served as a growth substrate, whereas a couple of similar other quaternary ammonium

alcohols also used in softeners did not. The isolated bacterium was identified by 16S-rDNA

sequencing as a strain of Pseudomonas putida with a difference of only one base pair to

P. putida DSM 291T• Despite their high similarity, the reference strain P. putida DSM 291T

was not able to grow with TM and the two strains differed even in shape when growing on the

same medium. This is the first microbial isolate able to degrade a quaternary ammonium

softener head group to completion. Previously described strains growing on quaternary

ammonium surfactants (decyl-trimethyl-ammonium, hexadecyl-trimethyl-ammonium and

didecyl-dimethyl-ammonium) either excreted metabolites or a consortium of bacteria was

required for complete degradation.

23

Chapter 2

INTRODUCTION

Today, mainly three structurally similar "esterquats", belonging to the group of cationic

surfactants, are used as laundry softeners in detergents (Figure 2.1). The worldwide annual

production of these surfactants probably exceeds 100'000 tons, with basically over 99 % of the

material being used in fabric care (Krueger et aI., 1998). The three "esterquats" appear to

hydrolyse rapidly (abiotically and/or biocatalysed) when reaching surface water or sewage

treatment plants, with the corresponding fatty acids and three quaternary ammonium alcohols

(QAAs) being the products (Giolando et aI., 1995; Hellberg et aI., 2000; Krueger et aI., 1998;

Puchta et aI., 1993; Simms et aI., 1992; Waters et al., 1991). The resulting QAAs are

trimethyl-2,3-dihydroxy-propyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and

methyl-triethanol-ammonium (MM) (Figure 2.1). The biodegradation properties of the parent

esterquat surfactant and the QAAs have been investigated in a variety of test procedures

Esterquat surfactants

oII

~c~~--O-C-R

I. ~H3C~I-C~-c~-0-C-- R

c~---c~-~ OH

MM-esterquat

lhydrolysis

f" fatty acids

lhydrolysis

f''''- fatty acids

Quaternary ammonium alcohols (QAAs)

o11

CH3 ~C~O--C-R

I. I ~~C~N-C~-CH-O-C~-R

ICH3

TM-esterquat

lhydrolysis

f'''- fatty acids

R: Fatty acid carbon chain derived from tallow, mainly C16 and C18

o11

~C~C~-O-C-R

I. ~H3C--~ ~C~-c~ -O-C- R

ICH3

DM-esterquat

CH3 ~C~OH

I. 1H3C - ~ ~-C~ ~- CH-~ OH

CH3 TM

H2T ~~- C~ -~~ OH

H C-~N·--CH -~C~ -OH3 I 2 • '2

CH3 OM

~ C--CH --OH• '2

1

2

H3c-i-c~-C~-OH

C~-~-OH MM

Choline, naturally related compound

CH3

I.H3C--i-- . C~--CH2-0H

C~

Figure 2.1. Structures of the three main commercially used esterquat surfactants, their hydrolysis

products and structure of the naturally related compound choline.

24


mimicking degradation in complex systems, for instance activated sewage treatment sludge.

Based on the performed tests, the parent esterquats and the QAAs are expected to be readily

and ultimately biodegradable (Giolando et aI., 1995; Krueger et aI., 1998; Matthijs et aI.,

1995; Puchta et aI., 1993; Simms et al., 1992; Waters et aI., 1991; Waters et aI., 2000).

The similarity of the QAAs and their structural relationship to the naturally occurring

compound choline (Figure 2.1) suggests that all three QAAs should degrade in a similar way

and with a similar rate, comparable to that of choline. However, despite their similarity they

show different degradation patterns as well as different degradation rates in GECD die-away

tests (Hales, 1998), and several choline-degraders, including one reference strain from DSM,

were not able to degrade any of the QAAs (own results). Also from the simple and choline

like structure of the molecule one would expect that many different microorganisms would be

able to utilise the compound as a single source of carbon, energy and nitrogen and that a

consortium would not be required for complete degradation. However, to date, no organisms

able to degrade these QAAs have been isolated and consequently the catabolic pathway for

neither of them has been elucidated yet. Considering the wide application of esterquat

surfactants as softeners in laundry detergents, we have therefore set out to enrich and isolate

microbes able to grow with these QAAs as their only source of carbon, energy and nitrogen.

Two different microorganisms able to grow with TM have been isolated and one strain,

designated TM 1, was selected for closer examination due to its high growth rate and

reproducible growth.

MATERIALS AND METHODS

Chemicals. The quaternary ammonium alcohols (QAA) (±)-2,3-dihydroxypropyl-trimethyl

ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium

(MM) were provided by Unilever (SEAC Safety and Environmental Assessment Center,

Unilever Research, Port Sunlight, UK) as the iodine salts. All other compounds were

purchased from Fluka, Buchs, Switzerland.

Isolation, growth and maintenance of organisms. For isolation of organisms (in batch and

continuous enrichment cultures), for growth tests and batch experiments, the following

25

Chapter 2

synthetic medium (SM) was used. It contained per litre of deionised water: MgS04'7HzO,

0.3 g; CaClz'2HzO, 0.02 g; NazHP04'2HzO, 2.05 g; KHZP04, 1.30 g; 1 ml of trace element

stock solution as described by Pfennig et al. (1981), but 3-times concentrated (containing per

litre: FeClz'4HzO, 4.5 g; MnClz'4HzO, 0.3 g; CoClz'6HzO, 0.36 g; ZnClz, 0.21 g;

CuClz'2HzO, 0.045 g; NazMo04'2HzO, 0.075 g; H3B03, 0.18 g; NiClz'6HzO, 0.075 g;

Na4EDTA'4HzO, 14.023 g); 1 ml of vitamin stock solution (which contained per litre:

pyridoxin'HCl, 100 mg; 50 mg of each, thiamine'HCI, riboflavin, nicotinic acid, D-Ca

pantothenic acid, p-amino benzoic acid, lipoic acid, nicotinamide, vitamin BIZ; biotin 20 mg

and folic acid 20 mg). The pH of the medium was always 7.0 except for experiments used for

the determination of pH optimum. For continuous cultivation the described medium was

slightly changed. Phosphate was exclusively added as KHZP04 (1.2 g rI) and the medium was

acidified with 0.2 ml r I of concentrated HZS04. The pH in the reactor was maintained at 7.0

by continuous addition of a NaOH/KOH (1 M/1 M) solution. Additionally, 0.1 ml r I of

antifoam (Silicon, emulsion 30 % in water, Fluka) was added to the medium to avoid

foaming. No vitamins were provided in continuous cultures and batches in bioreactors, since

isolate TM 1 was able to grow in the absence of vitamins.

The medium used for continuous cultivation was prepared as described above and it was

supplemented with the carbon source of choice after sterilisation by sterile filtration. For

sterilisation of batch media, MgS04'7HzO, CaClz·2HzO and trace elements were added to

nanopure water and autoclaved in Erlenmeyer flasks. Phosphate buffer, vitamins and the

carbon source of choice were added after sterilisation to the cooled-down medium by sterile

filtration. For substrate tests the complete medium was filtered sterile into heat-sterilised glass

test tubes. In each case, filtration was performed using sterile Millex-GP filters of 0.22 t-tm

pore size (Millipore, Volketswil, Switzerland).

If the growth substrate did not contain nitrogen, ammonia was added to the SM as N~CI,

0.8 g r I. For agar plates 1.5 % of highly purified agar (Biolife, Milano, Italy) was added to the

SM, both before sterilisation.

For experiments in complex liquid media, tryptic soy broth (TSB) (Biolife, Milano, Italy) was

used as a ten-fold dilution. Agar plates containing complex media of the appropriate tryptic

soy content were prepared by adding 13.5 g rI of highly purified agar to 4 g rI of tryptic soy

agar (TSA) (both Biolife, Milano, Italy).

26


Bacterial strains and storage. Pseudomonas putida DSM 291T was obtained from Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig,

Germany. For short-term storage all strains were plated on lO-fold diluted TSA or agar plates

containing SM and a selective carbon source. For long-term preservation all strains were

suspended in 30 % glycerol and stored at -80 QC. 16S-rDNA sequencing of strain TM 1 for

identification was performed by DSMZ.

Nutritional and biochemical properties. Utilisation of different carbon sources and selected

metabolic activities were investigated in various commercially available ready test kits such

as API 20 NE, API 50 CH (Biomerieux, Geneva, Switzerland) and Biolog GN plates (Biolog,

Hayward, California, USA) following the instructions of the manufacturer. For API 50 CH

and Biolog GN plates SM with N&CI (0.8 g r l) as nitrogen source was used. The utilisation

of selected carbon sources and combined carbon/nitrogen sources was tested in liquid culture

(5 ml in glass test tubes) at pH 7 using SM supplemented with the compound of choice (50

250 mg r l) by following turbidity. Controls without a carbon source were used. All tests were

inoculated with cells taken from colonies pregrown on tryptic soy agar. Cultures were

incubated at 30 QC in the dark and all tests were done in triplicates.

The Gram-reaction was tested with the classical staining procedure as described by Drews

(1968), the KOH method (Buck, 1982) and by testing for the presence of L-alanin

aminopeptidase (Bactident strips, Merck, Darmstadt, Germany).

Production of fluorescent pigments was tested by plating cells on agar containing an iron

deficient medium (Gould SI, containing per litre: Sucrose, 10 g; casamino acids, 5 g; glycerol

87 %, 10 ml; agar, 18 g; MgS04 1 M, 1.66 ml; CaCh 1 M, 0.18 ml).

SDS-PAGE of cellular proteins was performed as described by Laemmli (1970). Cells used

for SDS-PAGE were grown in batch cultures. Samples were taken in the exponential phase of

growth and harvested in Eppendorf tubes in a microcentrifuge (ALC, Milano, Italy). The

supernatant was discarded and the pellet stored at -20 QC not longer than one month prior to

SDS-PAGE.

Growth characteristics. Experiments for the determination of optimum growth conditions of

strain TM 1 were performed in batch cultures in Erlenmeyer flasks containing 100 ml of SM

27

Chapter 2

and TM (500 mg r l). Batch media were inoculated with cells pregrown in the same medium

taken from the exponential growth phase. Incubation was executed at different temperatures

(4, 12,20,25,28,30,32,37 QC) and pH (5.0; 6.0; 6.5; 7.0; 7.5; 8.0; 9.0). For batch growth at

different pH values the buffer stock solution was adjusted with either NaOH or HCI before

adding it to the medium. Growth was followed by measuring optical density at 546 nm

(ODS46) with a glass cuvette (1 cm) in a spectrophotometer (Kontron Uvikon, Zurich,

Switzerland). All experiments were performed at least in duplicate.

Determination of the maximum specific growth rate was performed for growth with TM at

optimum conditions following ODS46 under nutrient excess conditions in batch culture. For

this purpose, cells were transferred repeatedly into fresh medium before they had reached the

late exponential phase until they exhibited a stable JLmax'

Carbon and nitrogen balances were performed in batch and continuous culture both in a

bioreactor of 1 I working volume (Bioengineering AG, Wald, Switzerland) at 30 QC and pH

7.0. Chemostat cultures were run at a dilution rate (D) of 0.1 h- I and with a TM concentration

in the feed medium of 2 g r l. For DOC, TM, NHt, N02-, N03-, TMA, and Ntot analysis

samples were taken from the cultures and filtered (Millipore PVDF, 0.22 JLm membrane,

Millipore, Volketswil, Switzerland) using a vacuum pump and filtrates were stored at -20 QC

prior to analysis. Cell dry weight was measured by collecting cells from a known culture

volume on preweighed Nuclepore polycarbonate filters, 0.2 JLm pore size (Sterico AG,

Dietikon, Switzerland). Filters were washed once with distilled water and dried at 120 QC.

Dissolved organic carbon (DOC) was measured with a Tocor 2 Carbon Analyzer (Maihak,

Hamburg, Germany). ~+-N was determined by the indophenol method as described by

Scheiner (1976). N02-, N03- and low molecular weight organic acids in growth medium were

analysed by ion exchange chromatography (IonPac ATC1 anion trap column, IonPac AGll

guard column, analytical IonPac ASll 4 mm column, ASRS-II 4 mm suppressor auto

regenerating mode, and CD20 conductivity detector, Dionex, Olten, Switzerland). Elution was

performed with a gradient of NaOH 0.5 mM to NaOH 27.5 mM in 10 minutes at a flow rate

of 1 ml·min- I. N02- and N03- were estimated additionally with analytical test strips (Merck,

Darmstadt, Germany, concentration range: N02-: 0.1-3 g rI, N03-: 10-500 mg r l). TMA was

determined as described by Shen (1988). Total nitrogen (Ntot) was photometrically determined

with a Nitrogen Cell Test (Merck). The C and N content of the cells were determined with a

28


CHNS-O analyser (Carlo Erba Instruments, Milano, Italy). Cells used for C and N analysis

were washed three times with distilled water and lyophilised prior to analysis. CO2 and O2

were analysed by gas chromatography (gas chromatograph type GC-8A, Shimadzu Co.,

Tokyo, Japan) equipped with two parallel packed columns packed with molecular sieve 5 A

80/100 and Porapack Q 80/100, respectively (both from Brechbtihler AG, Schlieren,

Switzerland), the carrier gas was helium, detection was achieved by thermal conductivity.

TM, DM, MM and choline were measured by ion pair chromatography as described by Weiss

(1991) for the determination of choline except that the eluent composition was changed to a

ratio of 98 % hexansulfonic acid (2 mM) 1 2 % acetonitril. Samples were filtered and diluted

ten times (concentration < 40 mg r l) prior to analysis. The detection limit for the QAAs in

distilled water was 4 mg r l. However, in growth medium a ten-fold dilution of all samples

was required to reduce matrix component effects and consequently the detection limit in

growth medium was only approximately 40 mg r I.

RESULTS

Enrichment, isolation and identification. Enrichment for TM-degrading microorganisms

was performed in batch and continuous culture at 30 QC and a pH of 7 using SM

supplemented with TM as the sole source of carbon, energy and nitrogen at a concentration

range of 50 to 250 mg r I. Continuous enrichment cultures were run in small chemostats

(100 ml volume) at D - 0.04 h- I and a feed concentration of TM of 150 mg r I. Both batch and

continuous cultures were inoculated with soil (Duebendorf, Switzerland), river water (river

Chriesbach, Duebendorf, Switzerland) or activated sludge from two wastewater treatment

plants (WWTP Duebendorf, Switzerland, and model wastewater treatment plant, SEAC,

Unilever Research, Port Sunlight, UK). Enrichment cultures showing positive growth

(turbidity) were diluted appropriately, plated on SM/TM agar plates and colonies of different

morphology were picked. In this way, two different strains growing on TM, originating from

continuous enrichment cultures, were isolated in pure culture. Strain TM 1 was selected for

further investigations due to its high maximum specific growth rate of 0.40 h- I during growth

on TM and its good handling properties for laboratory work. Strain TM 2 was growing much

slower on TM (/Lmax(TM) - 0.16 h- I) and only about 40 % of initially provided carbon (as TM)

29

Chapter 2

was used in batch cultivation, whereas TM 1 utilised 80-90 % of the initially supplied TM

carbon.

By sequencing the 16S-rDNA gene isolate TM 1 was identified as a strain of Pseudomonas

putida (in the following text referred to as P. putida TM 1). An alignment of the

P. putida TM 1 sequence to sequences in the databases of EMBL (European Molecular

Biology Laboratory) and RDP (Ribosomal Database Project, MAIDAK et. al. 1999) resulted

in a similarity of 99.9 % (best hit) with one base pair difference to strain P. putida DSM 291T.

The difference was located at position 1136 (E. coli nomenclature) of the 16 S-rDNA

sequence, with adenine in P. putida TM 1 and thymine in P. putida DSM 291 T. Affiliation of

P. putida TM 1 and DSM 291T to true fluorescent Pseudomonads was verified by plating

them on Gould SI iron-deficient medium, on which the production of green-fluorescent

pigments was detected for both strains.

Morphology of colonies and cells. The cells of P. putida TM 1 were motile and elliptical in

shape with variable size (0.5-0.9 x 1.2-2.4 /-Lm) and two or three polar flagella (Figure 2.2a,d).

All cells were morphologically identical within the size range mentioned. Aggregates of a few

up to about a hundred cells were formed while growing with TM as sole source of carbon and

nitrogen, typically in the initial batch growth phase at low cell densities. Separation of

clustered cells was observed later at higher cell densities. The morphology of strain P. putida

TM 1 is clearly different from that of cells of the reference strain P. putida DSM 291T, which

appeared as slim rods with parallel cell walls (Figure 2.2b). The KOH and the L-alanine

aminopeptidase method indicated a Gram-negative nature of strain TM 1, whereas the Gram

staining gave equivocal results. Confirmation of the Gram-negative cell wall was obtained by

electron microscopy (Figure 2.2c).

On agar plates containing MSITM, P. putida TM 1 formed irregular colonies with a clearly

defined border of a beige, shiny appearance (diameter < 0.5 mm) after one day of incubation

turning into brown, mat colonies (diameter - 1 mm) after the second day. Longer incubation

times lead to larger, volcano-like colonies of up to 2 mm diameter without changes in color.

On TSA plates the colonies were circular with clearly defined borders (diameter 1 mm) and

appeared dark brown and mat after one day of incubation. Longer incubation times did not

alter the shape or the color and only the size increased to about 4 mm after three days.

30


d

-.,'-- fill;

1,/1..,\,

\,

,,-

'" ,.,,~ ."....

Figure 2.2. Morphology of isolate P. putida TM 1 (a) and of the reference strain P. putida DSM 291 T

(b) seen in the light microscope. Both strains were pregrown on tryptic soy broth. Electron microscope

pictures of thin sectioned (c) and negatively stained (d) cells of P. putida TM 1.

Colonies of P. putida DSM 291 T growing on TSA plates were irregular with a distinct border

after one day of incubation and of light brown, mat color with a diameter of about 1.5 mm.

After 2 to 3 days colonies became larger (up to 5 mm of diameter) and fringed, without

changing color.

Nutritional and biochemical properties. For the determination of the nutritional and

biochemical properties of P. putida TM 1 a variety of selected organic compounds were tested

and the commercially available test systems API 20 NE, API 50 CH and Biolog GN plates

were used. API 20 NE and API 50 CH and some selected tests were executed as well with

P. putida DSM 291 T.

31

Chapter 2

In growth tests using liquid SM, P. putida TM 1 was found to grow exclusively with the QAA

TM, which was used for its isolation, whereas DM and MM did not support growth. Choline,

the natural structurally related compound to TM, and all metabolites of its degradation

pathway, namely betaine, dimethylglycine, sarcosine, and glycine (Kortstee, 1970) were

utilised for growth by P. putida TM 1. Out of the additionally tested CIN-containing

compounds, only ethanolamine supported growth whereas methylethanolamine,

methyldiethanolamine, dimethyl-2-propanolamine, triethanolamine, ethylendiamintetraacetic

acid, and nitrilotriacetic acid did not. Cz-compounds such as ethanol, acetate or glyoxylate as

well as the fatty acids propionate and octanoate served as growth substrates, too, whereas the

Cl-compounds formate, methanol, trimethylamine, dimethylamine and monomethylamine did

not support growth. Reference strain P. putida DSM 291 T failed to grow with the QAAs TM,

DM and MM even if an additional nitrogen source (NH4Cl) was added to the batch culture

medium. Except for TM, P. putida DSM 291 T showed the identical substrate utilisation

pattern as P. putida TM 1 did.

To test whether DM or MM can be cometabolised, the two quaternary amines (DM 350 mg rl

and MM 440 mg r\ respectively) were pulsed into a continuous culture of P. putida TM 1

growing on TM as sole source of carbon and nitrogen. However, neither DM nor MM was

used, and the two pulsed compounds were washed out following the theoretical washout

curve (Figure 2.3a,b). No toxic or inhibitory effects of DM and MM on growth with TM were

observed, indicating that the two quaternary ammonium alcohols do not interfere with the

uptake of TM.

The results of API 20 NE, designed for non-enteric bacteria, are shown in Table 2.1 and give

an impression of the basic metabolic abilities of P. putida TM 1. Although, according to the

manufacturer, this test should allow identification of isolates belonging to the genospecies

P. putida, strain TM 1 could not be identified as a member of P. putida by API 20 NE. The

reason for the failure to identify strain TM 1 was found in the test for the assimilation of

adipate. Whereas the isolated strain P. putida TM 1 was able to grow with adipate as carbon

source P. putida DSM 291 T was not.

32


3.0 300C

~

Cl

2.0 200 .sCD z..,.'" I

0 +0

..,.I

1.0 100 Z

<500

0.0 0

3.0 500d

400 CCl

2.0 .sCD 300..,.

Z'"0 +I

0..,.

200 I1.0 Z

100 :tI-

0.0 0

-1 0 1 2 3 4 5 6 7 8 9 10Time [h)

Cl

200 .sz

I

+ ..,.I

100 Z<5oo

Cl

200 .sz

I

+..,.I

100 Z<5oo

300

o300

<><> b<:to~~~

'()''C

.......... "i-.4t ••<> '0' .<>.~

<>

a<>.~<><>.

~c>v •

..•..... i·Q~<>.<>

~""

<>

M 0-2 0 2 4 6 8 10 12

Time [h)

0.0

3.0

2.0

3.0

2.0

CD..,.'"o

o1.0

CD..,.'"o

o1.0

Figure 2.3. Co-metabolic ability of P. putida TM 1 in the chemostat. A pulse of a) DM (350 mg r l),

b) MM (440 mg r l), c) ethanol (410 mg r l

), and d) TM (400 mg r l) was added to a continuous culture

of P. putida TM 1 growing with TM (2 g r l) as the only carbon, nitrogen and energy source,

D =0.1 h- I. Pulses were executed at time zero. OD546 (.), TM (0), DOC (<», N~+-N (L.),

theoretical washout curve (---).

Regarding the test results obtained with API 50 CH, which allows testing growth with 49

different carbohydrates, P. putida TM 1 exhibited very poor growth on all sugars. Only slight

growth was detected on 7 different sugars after up to 96 hours of incubation. Sugars

supporting growth of P. putida TM 1 and differences to P. putida DSM 291T are listed in

Table 2.1.

Biolog GN plates provide a substrate utilisation pattern of Gram-negative microorganisms by

testing activity (due to a pH-change) on 95 different carbon sources, including a variety of

carbohydrates, acids and amino acids. P. putida TM 1 showed metabolic activity on most

tested acids, whereas activity was poor on the tested carbohydrates (data not shown),

confirming the results obtained with API 50 CH and API 20 NE test kits.

33

Chapter 2

Table 2.1. Physiological and morphological properties of P. putida TM 1 as collected with API 20

NE, API 50 CH and selected additional tests. If carbon source was not nitrogen-containing, N14CI

was added to the growth medium (0.8 g.-I). Note: Growth with arabinose was positive for both strains

when API 20 NE was used whereas P. putida DSM 291T exhibited no growth in test kit API 50 CH.

This difference may be caused by the different media used and the different concentrations of

provided substrate in these two tests and it points to the limitation of their interpretation. +...positive

reaction, definite growth; ± ...slight growth; -...no reaction, no growth; ( )...result assessed after 48

hours of incubation.

+ +-(+) -(+)-(±) -(±)-(±) -(±)-(±) -(±)-(±) -(±)

+ ++ +++ ++ ++ +

Properties tested

Shape:Motility:

API20NE:Reduction of nitrate to nitriteReduction of nitrates to nitrogenIndole productionAcidificationArginine dihydrolaseUreaseHydrolysis of B-glucosidaseProtein hydrolysisPresence of B-galactosidaseAssimilation of:

GlucoseArabinoseMannoseMannitolN-Acetyl-glucosamineMaltoseGluconateCaprateAdipateMalateCitratePhenyl-acetate

Presence of cytochrome oxidase

P. putida TM 1

ellipsoidmotile

+

P. putida DSM 291T

rodmotile

+

API 50 CH:GlycerolL-ArabinoseRiboseD-GlucoseD-FructoseGluconate2-Keto-gluconateAll 42 other sugars

±±±± +± ±± +± ±

Assimilation of:TMDMMMCholine

+

+

34

+


A comparison of protein patterns with SDS-PAGE was performed for both strains, P. putida

TM 1 and P. putida DSM 291T. P. putida TM 1 cells grown with TM or choline as sole

source of carbon and nitrogen exhibited different protein patterns suggesting that different

enzymes are involved in degradation of TM and choline despite their structural similarity

(Figure 2.4). Striking in the SDS-PAGE from both, TM- and choline-grown cells, was the

distinct protein band at about 68 kDa which was less pronounced in TSB-grown cells

(Figure 2.4). SDS-PAGE with cells of P. putida TM 1 and P. putida DSM 291T grown on

identical medium with same carbon source, namely either choline or TSA, resulted in

identical protein patterns for both substrates used (data not shown).

grown on grown on grown onTM choline TSB

kDaStandardHAA401

TM 1 TM 1 TMl

200.0

97.4

68.0 -

43.0 -

18.4 -

14.3 -

Figure 2.4. SDS-PAGE of total cellular proteins obtained from P. putida TM 1 grown with TM,

choline, and TSB. Arrows point out major differences in cellular protein between TM-grown and

choline-grown cells.

Growth characteristics. Optimum growth conditions of P. putida TM 1, growing with TM

as sole source of carbon and nitrogen, were found to be 30 QC and pH 6.5 to 8 resulting in a

f.1max of 0.40 ± 0.02 h-1. Growth was still observed at 4 QC, but not at 37 QC. At pH 9 the

specific growth rate was reduced to about 75 % of f.1max measured at optimum conditions,

whereas at pH 5 even a reduction to 20 % was observed. The C- and N-content of

35

Chapter 2

P. putida TM 1 biomass grown with TM was 45 % C and 12 % N of cell dry weight, which is

within the limits normally observed (Egli, 2000).

The ability of isolate TM 1 to degrade TM was found to be stable since cells grown on TSB

for more than one week (transferred to new medium every day, totally at least 60 generations)

were able to grow immediately with TM after transferring them back to TM-containing

medium again.

Typical batch growth of P. putida TM 1 with TM in synthetic medium is shown in Figure 2.5.

The culture grew exponentially in batch cultures. However, after reaching about 60 to 70 % of

final ODs46 an immediate decrease to a new constant growth rate of about 50 to 60 % of the

initial growth rate was observed. Excess nitrogen from TM was released in the form of

ammonia and the release was found to be proportional to the biomass increase. DOC and TM

decreased proportionally to the increase of biomass indicating that the growth yield during the

entire batch growth stayed constant.

0.8 600 6

500 5

0.6 -------------~-

400 ~=- 4 ~

Cl

oS Clco oS'<ton 0.4 300 ()0 3 z0 0 I

0 +'<t

~:r:

200 I- 2 z

0.2 -~.~.-. __._--

100

0.0 0 00 2 4 6 8 10 12 14

Time fhl

Figure 2.5. Batch growth of P. putida TM 1 with TM as sole source of carbon, energy, and nitrogen.

T =30 QC, pH =7.0 ± 0.1; OD546 (.), TM (0), DOe (0), NH/-N (6.).

36


The carbon and nitrogen balances in batch and continuous culture are given in Table 2.2. In

batch culture the residual TM concentration after exponential growth was below the detection

limit, i.e. below 40 mg r l. The final DOC concentration typically reached about 25 mg r 1

carbon (not including carbon originating from EDTA in the SM). Growth with TM in batch

culture was characterised by a low growth yield YX/c of 0.73 mg dry weight per mg carbon,

indicating a high level of carbon combusted to CO2 (60 %). Unusually, pH decreased

significantly (ca. 0.5 units) during growth despite the buffering and although excretion of

ammonia occurred. Since no organic acids such as acetate, formate, oxalate, or other small

strong acids could be detected the pH decrease was probably due to the high amount of CO2

produced during growth.

Table 2.2. Carbon and nitrogen balances for P. putida TM 1 growing with TM as sole source of

carbon, energy and nitrogen in continuous and batch culture. Mean values ± standard deviation are

given. Values reported are based on at least 4 samples in continuous culture and at least 2 samples in

batch culture.

Carbon balance Continuous culture Batch culture

mgC'h,1 % mgc·r l %

Input: 118.2 ± 3.5 100 279.5 100

Output (total): 117.2 ± 5.8 99 279.5 ±l.l 100

Biomass 27.9 ± 0.9 24 85.3 ± 0.5 31

DOC 6.4 ± 0.2 5 25.7 ± 0.6 9

CO2 dissolved 2.1 2

CO2 80.8 ± 5.7 68 168.5 ± 0.8 60

Input-Output: 1.0 ± 6.8 1 0.01) 01)

Nitrogen balance Continuous culture Batch culture

mgN'h,1 % mgN·r l %

Input: 23.1 ± 0.7 100 54.1 100

Output (total): 22.1 ± 0.7 96 54.1 ± 0.6 100

Biomass 7.2 ± 0.2 31 21.5 ± 0.1 40NH4+ 14.7 ± 0.4 64 27.9 ± 0.4 52

DON 0.2 ± 0.5 1 4.7 ± 0.4 8

Input-Output: 1.0 ± 1.0 4 0.01) 01)

1) Total CO2 release and DON were calculated as difference of measured input minus measured outputparameters, hence input - output = 0

In carbon-limited continuous culture (2 g r 1 of TM corresponding to about 1.075 gC r\ the

steady-state residual TM concentration at D = 0.1 h-1 was below the detection limit of

37

Chapter 2

40 mg r 1. Residual DOC concentration in the culture amounted to 56 mg r 1 which is a rather

high level of residual carbon in a C-limited continuous culture indicating the excretion of

carbonaceous products. However, no small organic acids (acetate, formate, oxalate or others)

were detected in the culture liquid. The growth yield in chemostat culture YXlC was

determined as 0.54 mg dry weight per mg carbon. This is significantly lower (26 %) than the

yield measured in batch culture. The decreased growth yield in the chemostat might be due to

a high requirement of energy for maintenance at low dilution rates but this still remains to be

elucidated. In any case, the excretion of products (DOC) was too low to explain the measured

difference in growth yields.

The majority of surplus nitrogen from TM was released as N~+ both in batch (- 85 %) and in

continuous culture (- 98 %) and neither N02-, N03- nor trimethylamine was excreted by

P. putida TM 1. Residual DON in continuous culture was 1 % of totally provided nitrogen

(from TM) and in the range of the standard deviation. In batch culture, residual DON

amounted to about 8 % of the totally provided nitrogen, the majority probably being

contained in TM. Since detection limit for TM was 40 mg rI, it was not possible to

characterise/identify the remaining DON.

Because in nature and wastewater treatment plants mixed substrate conditions are the rule

rather than the exception growth of TM 1 was investigated in batch and C-limited continuous

cultures using mixtures of substrates, too. In batch culture, no diauxic growth of

P. putida TM 1 was observed when the bacterium was cultivated in media containing TM as

sole source of nitrogen using either acetate or ethanol as an additional carbon source. The

different substrates were utilised simultaneously and the release ofN~+ into the medium was

highly reduced due to the additional formation of biomass (data not shown). An increase of

the growth rate was observed for the combination of TMlacetate, whereas no change in

growth rate was detected for TMlethanol. When pulsing ethanol (400 mg r 1) into a

continuous culture of P. putida TM 1, growing with TM (input 2 g r 1) as sole source of

nitrogen, ethanol was used immediately resulting in an increase of biomass concentration and

a decrease of the concentration of~+ in the culture medium. As a reference experiment,

TM (400 mg r 1) was pulsed to the same continuous culture resulting in an immediate

formation of additional biomass and an increase of N~+ concentration in the medium

proportional to the increase of biomass (Figure 2.3c,d).

38


DISCUSSION

Quaternary ammonium surfactants (cationic surfactants) have now been used in fabric care

for more than thirty years with an estimated world production of some 350'000 tonnes per

year (Van Ginkel, 1995). Therefore, several studies concerning the environmental properties

of these compounds have been performed (summarised in: Callely et aI., 1977; Krueger et aI.,

1998; Van Ginkel, 1995). Most investigations have focused on the fate, toxicity and

biodegradability in natural or simulated natural systems such as OECD test procedures. A

limited number of studies on the isolation of bacteria able to degrade quaternary ammonium

surfactants have been reported so far (Dean-Raymond & Alexander, 1977; Nishihara et aI.,

2000; Van Ginkel et aI., 1992). Microorganisms were isolated from activated sewage sludge

or soil using the quaternary ammonium surfactants decyl-trimethyl-ammonium, hexadecyl

trimethyl-ammonium and didecyl-dimethyl-ammonium as sole source of carbon. However,

the degradation of these compounds was either incomplete (excretion of tri- or dimethylamine

into the growth medium) or restricted to a consortium of at least two different

microorganisms. None of the isolated bacteria were able to completely degrade the alkyl part

and the methylated nitrogen part. Based on these studies and other investigations on the

biodegradation of a series of anionic, non-ionic, amphoteric and cationic surfactants Van

Ginkel (1996) concluded that complete degradation of most surfactants has to be achieved by

consortia of microorganisms. He suggested that only a few surfactants, i. e. alkane

sulphonates, alkyl sulphates and alkylamines are completely degraded by single

microorganisms.

In contrast, the P. putida TM 1 strain described here was able to completely degrade both the

Cl groups as well as the N-linked alkyl part (i.e. not the fatty acid part) of TM. The nitrogen

contained in TM was used as nitrogen source and excess nitrogen was excreted in the

mineralised form of NH/. No trimethylamine was found in the culture medium. It appears

that the methyl groups in TM were not incorporated into biomass but rather oxidised to CO2.

This speculation is supported by the low yield and high percentage of produced CO2 during

growth with TM as well as by the failure of P. putida TM 1 to grow with Cl compounds.

In previous studies nitrogen was always added to the media as an~+ salt in addition to the

nitrogen supplemented with the quaternary ammonium compound. Hence, no selection

pressure was applied for microorganisms able to use the nitrogen provided with the

39

Chapter 2

quaternary ammonium surfactant. This was perhaps a reason for the failure of previous

studies to isolate bacterial strains able to degrade quaternary ammonium surfactants to

completion.

In the literature, several degradation mechanisms of alkyl-trimethyl-ammonium compounds

have been postulated (Callely et aI., 1977; Van Ginkel, 1995):

A) Oxidation at the free end of the alkyl chain followed by stepwise degradation via beta

oxidation resulting in betaine, a natural widespread compound utilised by a variety of

ffilcroorgamsms.

B) Progressive demethylation of the nitrogen centre, then splitting off the nitrogen from the

alkyl chain as ammonia. The alkyl chain finally undergoes again beta-oxidation (fatty acid

metabolism).

C) Cleavage of the N-Calkyl bond providing trimethylamine and the aldehyde of the alkyl

chain. Whereas the alkyl chain again is degraded via common beta-oxidation,

trimethylamine is degraded by methylotrophic organisms.

Mechanism C), initial cleavage of the N-Calkyl bond, was proposed to be a general strategy of

microorganisms to gain access to the alkyl chains of quaternary ammonium compounds (Van

Ginkel, 1996). However, no speculation on the mechanism(s) responsible for the degradation

of TM by P. putida TM 1 can be made based on the experiments performed so far.

Considering the structural similarity of TM to the naturally related compound choline, one

also could speculate that the degradation of TM might occur in a similar way to choline or

that it might be channelled into the choline pathway, since the ability to degrade choline is

widespread amongst microorganisms (Bater & Venables, 1977; Ikuta et al., 1977; Kiene,

1998; Kortstee, 1970; Ohta-Fukuyama et aI., 1980; Shieh, 1964). However, neither one of the

organisms that we have isolated nor the reference strain P. putida DSM 291T (also capable to

grow with choline) was able to degrade TM or one of the other QAAs. Therefore, degradation

of TM may not necessarily be associated with choline degradation.

The majority of the previously isolated bacteria found to attack quaternary ammomum

surfactants were assigned to the genus Pseudomonas (Dean-Raymond & Alexander, 1977;

Nishihara et al., 2000; Van Ginkel, 1996; Van Ginkel et aI., 1992). Our strain was a true

fluorescent pseudomonad according to the current taxonomic definition (De Vos & De Ley,

1983). Despite the striking similarity of the isolated strain P. putida TM 1 to P. putida

40


DSM 291T based on 16S-rDNA sequencing (99.9 % corresponding to one different base pair)

P. putida TM 1 differs in substrate range and even in shape from the reference strain. This

demonstrates that ribosomal, genetic analysis is able to provide phylogenetic relationship,

indeed, but does not necessarily supply information on the specific metabolic ability of

isolates.

Further research will be focussed on the enzymatic degradation pathway of TM. This

information will be important with respect to the environmental behavior of these compounds

which may help in designing more biodegradable alternative quaternary ammonium

compounds.

ACKNOWLEDGEMENTS

We thank Dr. Ernst Wehrli, Laboratory for Electron Microscopy, ETHZ, for preparation of

the electron micrographs of strain P. putida TM 1 and I. Holderegger for CHNS-analysis.

41

Seite Leer /Blank leaf

QAA degrading bacteria

3. Isolation and characterisation of bacteria able to grow with quaternary

ammonium alcohols as sole source of carbon, energy and nitrogen

ABSTRACT

The quaternary ammOnIum alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium

(TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are

hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in

fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon,

energy and nitrogen. The strains were compared with a previously isolated TM-degrading

bacterium, which was identified as a representative of the species Pseudomonas putida

(Kaech & Egli, 2001). Two bacteria were isolated with DM, referred to as strains DM 1 and

DM 2, respectively. Based on 16S-rDNA analysis, they provided 97 % (DM 1) and 98 %

(DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both

strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is

typical for representatives of the genus Zoogloea. Using MM we isolated a gram-negative,

non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium

revealed 94 % identities (best hit) to Rhodobacter sphaeroides only. The strains MM 1 and

DM 1 were able to grow exclusively with the QAA used for their isolation. DM 2 was also

utilising TM as sole source of carbon, energy and nitrogen. However, all of the isolated

bacteria were able to grow with the natural and structurally related compound choline.

43

Chapter 3

INTRODUCTION

Today, esterquat surfactants are used in large quantities as softeners in washing detergents.

The annual production of esterquats probably exceeds 100'000 tons worldwide with basically

over 99 % of the material applied in fabric care (Krueger et al., 1998). The three mainly used

esterquat surfactants are shown in Figure 3.1. They hydrolise rapidly, abiotically and/or

biocatalysed, when reaching surface water or sewage treatment plants. The products are the

corresponding fatty acids and quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl

trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol

ammonium (MM) as displayed in Figure 3.1 (Giolando et al., 1995; Hellberg et al., 2000;

Krueger et al., 1998; Puchta et al., 1993; Simms et al., 1992; Waters et al., 1991). The parent

esterquats and the QAAs have been investigated extensively in standard biodegradation tests

Esterquat surfactants

o

oJlR

"" +/'---/ 0/N~OJlR

DM-esterquat

hydrolysis

\fatty acid

hydrolysis

\fatty acid

Quaternary ammoniumalcohols (QAAs)

oJl hydrolysis HO__ -"" /,---/OH

HO /'---/0 R~ ~ 'N+~N+ 0 '\ /~

/ ~OJlR fatty acid MM OHMM-esterquat

Choline, naturally related compound

R: Fatty acid carbon chain derived from tallow, mainly Cl6 and C18

Figure 3.1. Structures of the three main commercially used esterquat surfactants, their hydrolysis

products, and the structure of the naturally related compound choline.

44


(GECD, 1981) mimicking degradation in complex systems. Based on these tests, both, the

parent esterquats and the QAAs, are expected to be readily and ultimately biodegradable in

the environment (Giolando et a!., 1995; Krueger et a!., 1998; Matthijs et a!', 1995; Puchta et

al., 1993; Simms et al., 1992; Waters et al., 1991; Waters et al., 2000). Their structural

resemblance to the naturally widely occurring compound choline (Figure 3.1) suggests that

QAAs are degraded in a similar way and at rates comparable to that of choline. However, the

three QAAs showed very different degradation patterns and different degradation rates in

GECD die-away tests (Hales, 1998) despite their structural similarity. From the simple and

choline-like structure of QAAs one would also expect that many different microorganisms

would be able to utilise the compounds as sole source of carbon, energy and nitrogen and that

no consortium is required for their degradation. However, no choline degraders, including

reference strains from the German Culture Collection DSMZ (Deutsche Sammlung fUr

Mikroorganismen und Zellkulturen), were able to metabolise these QAAs (own results).

Moreover, up to now, no microorganisms degrading the three QAAs have been isolated and

consequently the catabolic pathways are not elucidated yet. In view of the widespread

application of the esterquats in laundry detergents, we have set out to isolate and enrich

strains able to grow with TM, DM and MM.

Here we report the isolation and characterisation of bacterial strains able to grow with the

QAAs DM and MM as sole source of carbon, energy and nitrogen. With DM two strains were

isolated, referred to as strain DM 1 and DM 2. Only one strain, capable to degrade MM,

designated as strain MM 1, was isolated. The isolate able to grow with TM (P. putida TM 1)

has been described previously in detail (Kaech & Egli, 2001).


Chemicals. The quaternary ammonium alcohols (QAAs) (±)-2,3-dihydroxypropyl-trimethyl

ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium

(MM) were provided by Unilever (SEAC Safety and Environmental Assessment Center,

Unilever Research, Port Sunlight, UK) as the iodine salts. All other chemicals were purchased

from Fluka unless indicated.

45

Chapter 3

Isolation, growth and maintenance of organisms. Isolation, growth and maintenance of

organisms was performed as described by Kaech & Egli (2001).

Bacterial strains and storage. Zoogloea ramigera Itzigsohn 1868AL (ATCC 19623, I-16-M)

was obtained from the German Culture Collection (DSMZ GmbH, Braunschweig, Germany).

For short-term storage, all strains were plated on lO-fold diluted TSA or agar plates

containing SM and a selective carbon source. For long-term preservation all strains were

suspended in 30 % glycerol and stored at -80°C.

16S-rDNA analysis. 16S-rDNA sequencing was executed as follows: 16S-rDNA fragments

of the isolated strains were amplified by PCR. The primers used for amplification were

originally reported by Weisburg et at. (1991). They were slightly modified resulting in the

sequencies 6F (5'-GGAGAGTTAGATCCTGGCTCAG-3') and 1510R (5'-GTGCTGCAGG

GTTACCTTGTTACGACT-3'). Both fragments were recovered and purified from the PCR

mixture by using Qiaquick spin columns (Qiagen, Basel, Switzerland), and ligated to pGEM

T-Easy (Promega, Wallisellen, Switzerland). Transformation of the ligation mixtures into

Escherichia coli DH5a resulted in several different recombinant strains, from which plasmid

DNAs were isolated according to Sambrook et at. (1989). Plasmid inserts of the proper size

were sequenced on both strands by using a Thermosequenase Kit (Amersham, Little Chalfont,

UK) with IRD-800 labelled primers (MWG Biotech, Ebersberg, Germany). Sequence

transcripts were separated and analysed on a LiCOR 4000L automated DNA sequencer

(LiCOR, Lincoln, NE, USA). All sequences were compared to the EMBL data bank entries

(European Molecular Biology Laboratory, Heidelberg, Germany) by using the BLAST2

routine (Gish, 1996-1999).

Nutritional and biochemical properties. Utilisation of different carbon sources and selected

metabolic activities were investigated in various commercially available ready test kits such

as API 20 NE and API 50 CH (Biomerieux, Geneva, Switzerland) following the instructions

of the manufacturer. For API 50 CH SM was used, amended with N~Cl (0.8 g r l) as

nitrogen source. The utilisation of selected carbon compounds and combined carbon/nitrogen

sources was tested in liquid culture (5 ml in glass test tubes) at pH 7 by following turbidity

46


using SM supplemented with the compound of choice (50-250 mg r 1). Controls without a

carbon source were used. All tests were inoculated with cells taken from colonies pregrown

on tryptic soy agar. Cultures were incubated at 30 QC in the dark and all tests were done in

triplicates.

The Gram-reaction was tested with the classical staining procedure as described by Drews

(1968), the KOH method (Buck, 1982) and by testing for the presence of L-alanin

aminopeptidase (Bactident strips, Merck, Darmstadt, Germany).

SDS-PAGE of cellular proteins was performed as described by Laemmli (1970). Cells used

for SDS-PAGE were grown in batch cultures. Samples were collected in the exponential

phase of growth and cells were harvested by centrifugation in Eppendorf tubes in a

microcentrifuge (ALC, Milano, Italy). The supernatant was discarded and the pellet stored at

-20 QC not longer than one month prior to SDS-PAGE.

Growth characteristics. Experiments for the determination of optimum growth conditions of

the strains were performed in batch cultures in Erlenmeyer flasks containing 100 ml of SM

and the carbon source of choice (500 mg r1). Batch media were inoculated with cells pre

grown in the same medium taken from the exponential growth phase. Incubation was done at

different temperatures and pH. For batch growth at different pH values (5, 7, 9) the pH of the

buffer stock solution was adjusted with either NaOH or HCI before adding it to the medium.

With the exception of DM 1, growth was followed by measuring optical density at 546 nm

(ODS46) with a glass cuvette (1 cm) in a spectrophotometer (Uvikon Kontron, ZUrich,

Switzerland). Strain DM 1 formed huge flocs during growth, therefore, biomass was followed

by measuring excreted N~+, which turned out to be proportional to biomass formation. This

was confirmed by comparing the amount of cellular protein formed (measured with the Bio

Rad protein assay, Bio-Rad Laboratories GmbH, Munich, Germany) with the N~+ excreted

during batch cultivations. All experiments were performed at least in duplicate; numbers

reported with standard deviations always from triplicates.

Determination of the maximum specific growth rate (/lmax) was performed using optimum

conditions and under nutrient excess in batch culture following ODS46 and~+ excretion,

respectively. For this purpose, cells were transferred repeatedly into fresh medium before they

had reached the late exponential phase until they exhibited a stable /lmax.

47

Chapter 3

Carbon and nitrogen balances were performed in batch culture using bioreactors of 1 I

working volume (Bioengineering AG, Wald, Switzerland) or Erlenmeyer flasks of 2 I volume

under optimum conditions. The concentrations of DM and MM used was in the range of 200

to 300 mg r I. Balances for the strains DM 1, DM 2 and MM 1 from two independent cultures

were calculated over the entire growth phase, whereas mean values of parameters (formed

biomass, C- and N-content of the cells, DOC, N~+) were based on at least triplicates

obtained from independent batch experiments. Samples were taken from the culture,

centrifuged at 15000*g in a microcentrifuge (ALC, Milano, Italy) for 5 min and the

supematant was either directly used or stored at -20°C prior to analysis of the following

parameters: DOC, DM, MM, N~+, N02- and N03-. Cell dry weight was measured by

collecting cells from a known culture volume on preweighed Nuclepore polycarbonate filters

of 0.2 /lm pore size (Sterico AG, Dietikon, Switzerland). Filters were washed once with

distilled water and dried at 120°C. DOC was measured with a Tocor 2 Carbon Analyzer

(Maihak, Hamburg, Germany). Carbon originating from EDTA in the medium was subtracted

from DOC for the carbon balance. N~+-N was determined by the indophenol method as

described by Scheiner (1976). N02-, N03- and low molecular weight organic acids in growth

medium were analysed by ion exchange chromatography (IonPac ATC1 anion trap column,

IonPac AGl1 guard column, analytical IonPac ASl1 4 mm column, ASRS-II 4 mm

suppressor auto-regenerating mode, and CD20 conductivity detector, Dionex, Olten,

Switzerland). Elution was performed with a gradient of NaOH 0.5 mM to NaOH 27.5 mM in

10 minutes at a flow rate of 1 ml min- I. N02- and N03- were estimated additionally with

analytical test strips (concentration range: N02-: 0.1-3 g rI, N03-: 10-500 mg rI, Merck,

Darmstadt, Germany). The C and N content of the cells was determined with a CHNS-O

analyser (Carlo Erba Instruments, Milano, Italy). Cells used for C and N analysis were

washed three times with distilled water and lyophilised prior to analysis. DM and MM were

measured by ion pair chromatography as described earlier for the determination of choline

(Weiss, 1991), except that the eluent composition was changed to a ratio of 98 %

hexansulfonic acid (2 mM) I 2 % acetonitrile. Samples were filtered and diluted ten times

(concentration < 40 mg r I) prior to analysis. The detection limit for the QAAs in distilled

water was 4 mg r I. However, in growth medium a ten-fold dilution of all samples was

48


required to reduce matrix component effects and consequently the detection limit in growth

medium was only approximately 40 mg r l.

RESULTS

Enrichment, isolation and identification. Enrichment for QAA degrading microorganisms

was performed in batch and continuous culture at 30 QC and a pH of 7. SM was used,

supplemented with either DM or MM as the sole source of carbon, energy and nitrogen at a

concentration range of 50 to 250 mg r l. Continuous enrichment cultures were run in small

chemostats (100 ml working volume) at D - 0.04 h- l and a feed concentration of the substrate

of 200 mg r l. Both batch and continuous cultures were inoculated with soil (Duebendorf,

Switzerland), river water (river Chriesbach, Duebendorf, Switzerland) or activated sludge

from two wastewater treatment plants (WWTP Duebendorf, Switzerland, and model

wastewater treatment plant, SEAC, Unilever Research, Port Sunlight, UK). Enrichment

cultures showing turbidity were diluted appropriately, plated on selective SMlQAA agar

plates and colonies of different morphology were picked. In this way, three different strains

originating from continuous enrichment cultures were isolated in pure culture, two of them

using DM (strains DM 1 and DM 2) and one strain using MM (referred to as strain MM 1) as

the sole source of carbon, energy and nitrogen.

The strains DM 1, DM 2 and MM 1 were identified by sequencing the 16S-rDNA gene. For

DM 1 (EMBL: AJ440749) and DM 2 (EMBL: AJ440750) the alignment to the sequences in

the EMBL database (European Molecular Biology Laboratory, Heidelberg, Germany) by

using the BLAST2 routine (Gish, 1996-1999) provided identities (best hit, total 1448 base

pairs) to Zoogloea ramigera Itzigsohn 1868AL (EMBL: X74915) of 97 % and 98 %,

respectively. Best hit for the alignment of the MM 1 sequence (EMBL: AJ440751) was found

to be Rhodobacter sphaeroides (EMBL: X53854) with identities of 94 % (total 1434 base

pairs) only.

Morphology of cells and colonies. The shape, size and flagellation of the cells of strain

DM 1 and DM 2 was very similar. They were long, slim rods with a size of 0.6-0.9 x 2.0-3.5

p,m (Figure 3.2a,b). Both microorganisms were motile with one to several polar flagella. The

49

Chapter 3

attachment of the flagella was not at the polar end of the rod-shaped cells, but slightly shifted

to the long side (Figure 3.2b). On average, DM 2 cells were slightly longer and slimmer than

the cells of DM 1. While growing with DM, the cells of both DM 1 and DM 2 formed

inclusion bodies, pointing to the accumulation of polyhydroxyalkanoates (PHA) (Figure

3.2a). Cells of DM 1 formed huge aggregates (Figure 3.2c) during growth with all tested

substrates (see nutritional and biochemical properties). The aggregates often stuck loosely to

the sides of silicon tubes used for sampling in batch cultures. However, DM 1 did not possess

a visible zoogloeal, gelatinous matrix in the light microscope. Even after staining with 1 % of

crystal violet as described by Friedman & Dugan (1968), no exocellular material was evident.

Figure 3.2. Electron micrographs of thin-sectioned cells (a) and negatively-stained cells (b) of strain

DM 1. Arrow points to a storage granule, probably PHA. Circles point out the attachment sites of the

flagella of strain DM 1. Cells of isolate DM 2 (not shown) looked the same as those taken of DM 1,

except that the cells were slightly thinner and longer. c) Aggregates of DM 1 cells formed during batch

growth, as seen in the light microscope.

50


In contrast, cells of DM 2 did not form any aggregation or flocs independent on the growth

substrate and cell density. Obviously, no matrices or floc-forming exopolysaccharides were

produced by this strain.

On agar plates containing TSA (10 % tryptic soy content), the colonies of strain DM 1 were

circular with a clearly defined border, and still very small after two days of incubation at

30 QC (diameter 1 mm). Colonies appeared light brown and slightly shiny. After six days, the

colonies reached a diameter of 2 mm. The border changed to slightly irregular shape and the

colonies turned brown with a narrow, bright rim. The entire colonies were movable when

pushed with an inoculation loop. If DM 1 was plated on SM/DM plates, the colonies were

white and shiny and reached a diameter of 1 mm at the most. Movability of the entire colonies

was detected on these plates, too.

Colonies of DM 2, grown on TSA plates (10 % tryptic soy content), showed the same

appearance as those of DM 1 after two days of incubation. In contrast to DM 1, DM 2

colonies were dark brown in the center of the colony and became continuously lighter towards

the border after six days of incubation. The maximum diameter after this time was 2 mm.

DM 2 colonies, grown on SM/DM plates, showed the same appearance as DM 1. In contrast

to DM 1, it was not possible to move them as a whole on the agar surfaces.

The type strain Z ramigera Itzigsohn 1868AL was plated on TSA for colony description, too.

The size of the colonies was identical to DM 1 and DM 2. After two days, the colonies were

1 mm in diameter and reached 2 mm after an incubation time of six days. The colonies after 2

days were circular, volcano-like, mat and of light brown colour with a dark brown, clearly

defined border. After six days of incubation, colonies turned dark brown in the centre with a

continuous gradient to a light brown border and the whole colonies were sprinkled with small,

dark dots. Movability of the entire colonies on agar plates was also observed. Since the type

strain Z ramigera Itzigsohn 1868AL did not grow on SM/DM plates, no colony description

can be made for this case.

The cells of isolate MM 1 were non-motile and without flagella. They were ovoid to rod

shaped and the size was 0.6-1.1 x 1.3-2.0 /lm (Figure 3.3a). MM 1 did not form any

aggregates during growth on all tested substrates. High amounts of inclusions bodies were

observed while growing with acetate (Figure 3.3b), whereas only few inclusions were found

51

Chapter 3

b

10/lm

Figure 3.3. a) Electron micrographs of thin-sectioned cells of strain MM 1. Anows point to storage

granules, probably PHA. b) Storage granules in cells of strain MM 1 (anows) grown with acetate, as

seen in the light microscope.

in cells growing with MM. Fluorescent staining of the inclusions with nile red pointed to

accumulation of a lipophilic storage compound, probably PHA.

Strain MM 1 showed poor growth on TSA plates (10 % tryptic soy content). When streaked

out on agar plates, best growth was observed in the region of the initially highest

concentration of the cells where a continuous film of cells developed. Only a few single

colonies were found. Single colonies reached a diameter of about 1 mm after three days of

incubation. They appeared as light brown, mat colonies with a clearly defined border and a

rough looking surface. After nine days, the colonies reached a diameter of about 2-3 mm in

diameter. The border turned irregular and the colonies were sprinkled with dark dots.

Based on the 16S-rDNA analysis all isolates belong to the group of Gram-negative microbes.

This was found as well with the L-alanine aminopeptidase method. Whereas the Gram

staining confirmed this result for isolates DM 1 and DM 2, equivocal results were found for

MM 1. The KOH method did not show the formation of gluey material (i. e. indicating a

Gram-negative cell wall) for either of the strains. For all isolates, confirmation of a Gram

negative cell wall was obtained by electron microscopy.

Nutritional and biochemical properties. For the determination of the nutritional and

biochemical properties of the isolated strains a variety of selected organic compounds was

tested and the commercially available test systems API 20 NE and API 50 CH were used. API

20 NE and some selected tests were executed as well with the reference strain Z. ramigera

Itzigsohn 1868AL.

52


In growth tests using liquid SM and TM, DM or MM as the sole source of carbon, energy and

nitrogen, DM 1 and MM 1 were found to grow exclusively with the QAA used for their

isolation (DM and MM, respectively). In contrast, strain DM 2 was able to grow with DM as

well as with TM. Choline, the natural structurally related compound to the QAAs, and the

typical metabolites of its catabolism, i. e. betaine, dimethylglycine and sarcosine (Kortstee,

1970), were utilised for growth by all isolated strains. Glycine was used as growth substrate

by DM 1 and DM 2, but not by MM 1. From the additionally tested CIN-containing

compounds, ethanolamine served as growth substrate for all isolated strains, but only MM 1

was able to grow with methylethanolamine, too. Methyldiethanolamine, dimethyl-2

propanolamine, triethanolamine, ethylendiamintetraacetic acid, and nitrilotriacetic acid did

not support growth for any of the isolated strains. With the C2-compound ethanol, growth was

detected for DM 1 and DM 2, but not for MM 1. On the other hand, the C2-compound

glyoxylate supported growth for DM 1 and MM 1, but not for DM 2. Acetate, and the fatty

acids propionate and octanoate served as growth substrate for all three strains. The C1

compounds formate, methanol and monomethylamine did not support growth for any of the

isolated bacteria.

The tested reference organism Z. ramigera Itzigsohn 1868AL did not grow with TM, DM or

MM. With the naturally related compound choline, however, growth was detected. Since the

most closely related strain to MM 1 (based on 16S-rDNA sequence) was Rhodobacter

sphaeroides, MM 1 was also tested for anoxic, phototrophic growth which is found for all

representatives of the genus Rhodobacter (Dworkin et al., 1999-2002). However, MM 1 was

not able to grow under such conditions. Neither growth nor production of pigments was

observed with MM 1 incubated anaerobically in the light with acetate as the source of carbon.

Table 3.1 shows the results of test API 20 NE designed for identification of non-enteric

bacteria. The test gives an impression of the basic metabolic abilities of the different

microbes. However, the pattern did not allow an identification of the isolated strains.

Nevertheless, the test showed differences among all the isolated and the reference strain and,

therefore, allowed distinguishing between them.

53

Chapter 3

Table 3.1. Physiological and morphological properties of strains DM 1, DM 2, MM 1 and Z. ramigera

Itzigsohn 1868AL as collected with API 20 NE and other selected tests. If the carbon source supplied

for growth in assimilation tests was not containing nitrogen, NI4CI was added to the growth medium

(0.8 g r'). +...positive reaction, definite growth; ± ... slight growth; -...no reaction, no growth; nd ...not

determined.

Properties tested Isolate DM 1 Isolate DM 2 Z. ramigera Strain MM 1Itzigsohn 1868AL

Shape: rod rod rod rodMotility: motile motile motile non-motile

API20NE:Reduction of nitrate to nitrite + +Reduction of nitrates to nitrogenIndole productionAcidificationArginine dihydrolaseUrease +Presence of B-glucosidase + + + +Protein hydrolysisPresence of B-galactosidase ± + + +Assimilation of:

Glucose + + +Arabinose + + +Mannose + +Mannitol + +N-Acetyl-glucosamine + +Maltose + + +GluconateCaprateAdipateMalate +CitratePhenyl-acetate

Presence of cytochrome oxidase + nd

Growth with:TM +DM + +MM +Choline + + + +

54


API 50 CH, which allows testing for growth with 49 different carbohydrates, provided the

following results for the isolated microorganisms. MM 1 did not grow on any of the sugars.

Slight growth of DM 2 was detected with 7 sugars, whereas DM 1 showed definite growth on

26 different sugars.

SDS-PAGE of the isolated strains, including P. putida TM 1, grown with the QAAs, provided

clearly different protein patterns (data not shown). No SDS-PAGE was performed with the

reference strain Z. ramigera Itzigsohn 1868AL because this strain was not able to grow with

any of the QAAs.

Since MM 1 showed 94 % identities only to Rhodobacter sphaeroides at the 16S-rDNA level

and did not exhibit the key properties found in all strains of Rhodobacter, no further

comparison was made using Rhodobacter sphaeroides.

Growth characteristics. Optimum growth conditions for isolate DM 1 growing with DM as

sole source of carbon, energy and nitrogen turned out to be 30 QC and a pH of 7, and resulted

in a fJ-max of 0.073 ± 0.007 h-1• Growth was still detected at 4 QC, but not at 37 QC. At a pH of

9, the growth rate was reduced to about 60 % of fJ-max and at pH 5 cells did not grow anymore.

The content of carbon and nitrogen in the biomass was 50 ± 3 % and 13 ± 1 % of the cell dry

weight, respectively.

Floc formation of strain DM 1 did not allow reliable determination of growth by OD

measurements. However, in batch cultivation, strain DM 1 grew exponentially (Figure 3.4a,b)

measured by excretion of NH/. DOC decreased to about 57 ± 2 % of the carbon

concentration provided initially as DM. 24 ± 2 % of the carbon were incorporated into

biomass and the remainder (19 ± 3 %, calculated as difference to 100 %) was most probably

released as CO2 (Figure 3.5). Proportionally to the biomass formation surplus nitrogen was

excreted as~+ and it amounted finally to about 21 ± 1 % of initially provided nitrogen in

DM. No nitrate or nitrite was detected in the culture liquid, some 29 ± 3 % was found

incorporated into the biomass, and the remaining nitrogen (50 ± 3 %) was present in the

culture supernatant as dissolved organic nitrogen (Figure 3.5). Strain DM 1 metabolised not

all of the initially provided DM. At the end of batch cultures, about 35 % of the initially

provided DM was left untouched in the culture liquid (Figure 3.4a). The reason for this

behavior was not caused by a shift of the pH, since the pH was controlled and adjusted if

55

Chapter 3

necessary during batch growth. Moreover, different initial concentrations in the range of 250

to 500 mg r l of DM in batch cultures provided the same outcome, and in the same synthetic

medium strain DM 2 was able to produce about twice as much biomass than strain DM 1.

Therefore, the incomplete utilisation of DM in batch cultures of strain DM 1 was most likely

not caused by a limitation of nutrients or trace elements in the SM. Furthermore, using higher

concentrations of other CIN sources, strain DM 1 as well as other strains were able to produce

much more biomass in the SM used without any indication of a nutrient or trace element

limitation.

Unutilised DM contributed to about half of the final DOC concentration (Figure 3.4a) and

made up all of final DON. This indicates that only carbon-containing metabolites and no

organic, nitrogen-containing compounds were excreted during growth. However, these carbon

compounds have not been identified so far. The growth yield in batch cultures was

approximately 1.1 ± 0.1 g dry weight per mg of carbon.

For strain DM 2, optimum growth conditions with DM as the sole source of carbon, energy

and nitrogen were found to be 25 QC and a pH of 7 - 9. The maximum specific growth rate

J-tmax was 0.078 ± 0.005 h- l. Whereas cells of DM 2 exhibited still growth at 4 QC, no growth

was detected at 37 QC and below a pH of 5. The carbon and nitrogen content of the cells was

46 ± 3 % and 12 ± 1 % of the cell dry weight, respectively. DM 2 grew exponentially in batch

cultures (Figure 3Ac). At the end of the exponential growth phase only 4 ± 1 % of the carbon

initially provided as DM were left. 41 ± 2 % of the carbon were assimilated into biomass and

the remainder (55 ± 3 %, calculated as the difference to 100 %) was combusted to CO2

(Figure 3.5a). Surplus nitrogen was excreted into the medium as .NI4+ and excretion was

proportional to the biomass increase. The final amount of NH/-N excreted was 45 ± 2 % of

the nitrogen initially provided as DM and 55 ± 2 % of the nitrogen was fixed into the biomass

(Figure 3Ac, 3.5b). No organic nitrogen (0 ± 4 %) was detected in the culture liquid at the end

of the exponential growth phase. Chemical analysis of DM confirmed that in batch culture the

DM provided was degraded to completion by strain DM 2 with a yield of 0.93 ± 0.05 mg dry

weight per mg of carbon. While growing with the QAA TM, DM 2 reached a J-tmax of 0.10 ±

0.01 h- l, which was much slower than the J-tmax found for P. putida TM 1 of 0040 ± 0,02 h- l

(Kaech & Egli, 2001).

56


(a)

0.15 ..,..------------------r 300

<D~l!)

oo

0.10

0.05

+

+ +OM250

200 O'lE..........

150 ()oo

100 2o

50

3020Time [h]

10o0.00 +---~------.----.,.-----+0

40

(b)

0.15 --r----------------"'T"" 10

0.00 +--------,------.----....-----4

<D~l!)

oo

0.10

0.05

o 10 20Time [h]

30

8

6

4

2

o40

..--....,O'l.sz+'~

IZ

Figure 3.4. Batch growth of strains DM 1 (a, b), DM 2 (c, see following page) and MM 1 (d, see

following page) with DM (a, b, c) and MM (d) as sole source of carbon, energy and nitrogen.• DM,

o MM, 0 DOe, 0 OD546 mu, 6. NH/-N. Note: The detection limit of DM and MM with the

analytical method applied was about 4 mg r l. Since samples had to be diluted ten times before

analysis to reduce matrix effects, the conclusive detection limit raised to about 40 mg r l and probably

caused that the final DM level (zero) in Figure 3.4 c was reached before the beginning ofthe stationary

phase as detected by OD.

57

Chapter 3

(c)

0.35 300 20

0.30 250,........,

15,........,0.25

~

~

200 0>0>E

~ 0.20........ .s

0 150 g 10200.15 0 I

+'<t

100 ~I2

0.105

0.05 50

0.00 0 00 10 20 30 40

Time [h]

(d)0.5 300 5

0.4 0250 4

,........,~ ,........,

200 ~ ~

0>(0 0.3 E 3 0>'<t ........ EID

150 ()........

00 0 2

0 I

0.2 2 +'<t

100 ~ I~

2

0.1 50 1

0.0 0 0

0 2 4 6 8 10 12

Time [h]

58


Optimum growth for strain MM 1 during growth in mineral medium with MM as a sole

source of carbon, energy and nitrogen was observed at 37 QC and at a pH of 7. The maximum

specific growth rate J.Lmax was 0.205 ± 0.006 h-1. Slow growth in batch culture (- 0.006 h- 1

)

was also detected at a temperature of 4 QC. At 38 QC, the specific growth rate was reduced to

about 65 % of J.Lmax and at 39 QC no growth was detected anymore. The carbon and nitrogen

content of the cells was 44 ± 3 % and 11 ± 1 % of the cell dry weight, respectively. In batch

culture strain MM 1 grew exponentially with a constant specific growth rate until entering the

stationary phase (Figure 3.4d). A relatively high proportion of 31 ± 4 % of the initially

provided carbon from MM was left in the medium in an unidentified form when the culture

entered the stationary phase. 37 ± 1 % was incorporated into the biomass and 32 ± 5 %

(calculated as the difference to 100 %) was combusted to CO2 (Figure 3.5a). The nitrogen

balance for growth with MM in batch culture (equivalent to 100 %) gave the following results

at the end of the exponential growth phase: 57 ± 1 % was fixed in biomass, 42 ± 2 % was

found as organic nitrogen in the culture liquid (DON, calculated as the difference to 100 %)

and only 1 ± 1 % was excreted as NH/-N (Figure 3.5b). No other nitrogen compounds like

N02- or N03- were found in the culture broth and MM disappeared completely during batch

growth (Figure 3.4d). It seems very likely that the high amount of residual nitrogen as DON

was a dead-end metabolite(s), which cannot be used anymore for growth. This was confirmed

with the data from samples taken four hours after the cells had entered the stationary phase,

where the same amounts of carbon and nitrogen were detected as at the beginning of the

stationary phase. The ratio of carbon to organic nitrogen in the remainder was 4.7 but it is

uncertain whether or not it is a single compound or several transformation products. Attempts

to identify the compounds by ion-pair and anion chromatography were not successful. The

yield during batch growth was 1.21 ± 0.07 mg of dry weight per mg of carbon.

59

Chapter 3

(a)

80

60%

40

20

oDM 1 DM 2 MM 1 TM 1

Strain

~ Biomass 11DOe D cO2

(b)

100 ...---,.---...---,---r-.----

80

60%

40

20

oDM 1 DM 2 MM 1 TM 1

Strain

~Biomass IIDON DNH/

Figure 3.5. (a) Carbon and (b) nitrogen balances of strains DM 1, DM 2 and MM 1 obtained from

batch cultures. Balances of P. putida TM 1 are shown for comparison. Total CO2 and DON were

calculated as difference of experimentally measured input minus experimentally determined output

parameters. Standard deviations are given in the text.

60


DISCUSSION

Several studies on the environmental properties of quaternary ammOnIum compounds

(cationic surfactants) have been performed since they have been used for more than thirty

years in large quantities (Callely et al., 1977; Garcfa et al., 2000; Garcfa et al., 2001; Krueger

et al., 1998; Valles et al., 2000; Van Ginkel, 1995). However, most of these investigations

comprise data on the fate, toxicity and biodegradability in the environment and in simulated

natural systems. Only a limited number of studies reports on the isolation of bacteria able to

degrade quaternary ammonium compounds (Dean-Raymond & Alexander, 1977; Nishihara et

al., 2000; Van Ginkel et al., 1992). The compounds used for the isolation of microorganisms

from sewage treatment sludge were decyl-trimethyl-ammonium, hexadecyl-trimethyl

ammonium and didecyl-dimethyl-ammonium as sole source of carbon. These compounds

were either degraded incompletely with tri- or dimethylamines being excreted, or microbial

consortia were required for their complete mineralisation. None of the isolated bacteria was

able to degrade both, the alkyl based and the methylated nitrogen part of the molecule. Based

on a series of studies on anionic, non-ionic, amphoteric and cationic surfactants Van Ginkel

(1996) proposed that the complete degradation of most surfactants is performed by consortia

of microorganisms. He suggested, that only alkane sulfonates, alkyl sulphates and

alkylamines are entirely degraded by individual strains. The lack of the isolation of microbes

degrading quaternary ammonium surfactants to completion may be due to the fact that in all

those studies nitrogen was provided in the form of ammonium in addition to the nitrogen

containing surfactant. In this way, no selective pressure to use the nitrogen deriving from the

surfactant itself was imposed during enrichment and isolation.

In contrast, we report here the isolation of pure bacterial isolates that were able to grow with

the quaternary ammonium alcohols DM and MM as a sole source of carbon, energy and

nitrogen. Hence, all bacterial strains were able to degrade the carbon-containing chains (or at

least parts of it) as well as to use the nitrogen contained in the quaternary ammonium part of

the molecule for biomass formation. As an exception, strain DM 1 did not metabolise all of

the provided QAA DM in batch cultures. This behavior was not caused by a shift of the pH or

by a limitation of nutrients in the medium and therefore this phenomenon remained

unexplained. In contrast, DM 2 did completely degrade DM during batch cultivation with

only a small amount of residual DOC being left at the end of the growth and all surplus

61

Chapter 3

nitrogen released as ammonium. Strain MM 1 again metabolised all the provided QAA MM

in batch cultures but excreted high amounts of organic, nitrogen containing not yet identified

compounds that remained untouched in the stationary phase. The degradation of TM again

proceeded with a different pattern. Here, it was observed that P. putida TM 1 consumed all

the provided TM in batch culture and all surplus nitrogen was released as ammonium with a

considerable part of the initial carbon remaining as unidentified dissolved organic carbon

(Kaech & Egli, 2001). Obviously, each of the isolated strains degraded its QAA in a different

way despite the structural similarity of the QAAs. Even the two isolates DM 1 and DM 2,

which turned out to be closely related based on their 16S-rDNA sequence, showed a totally

different behavior during growth with the same QAA. Hence, one is tempted to speculate that

the degradation of the QAAs is achieved by different mechanisms. Surprisingly, DM 2 was

the only organism able to grow with more than one QAA. Considering the structural

similarity of the QAAs one would expect that microbial strains would be capable to degrade a

whole range of different quaternary ammonium alcohols. Obviously, this is not the case.

QAAs are structurally similar to choline. This compound is found ubiquitously in nature and

the ability to degrade choline is widespread amongst microorganisms (Kortstee, 1970;

Rosenstein et al., 1999; Shieh, 1964). Since all isolated bacterial strains were able to grow

with choline, one can speculate that the QAAs are metabolised in a similar manner as choline

is. However, neither of the bacteria we isolated with choline (results not shown) nor the

reference organisms Z. ramigera Itzigsohn 1868AL, nor P. putida DSM 291T

, both also able to

grow with choline, were able to degrade any of the QAAs. Therefore, the ability to degrade

choline does not go along with the ability to catabolise TM, DM or MM. Earlier, several

mechanisms have been proposed (Callely et aI., 1977; Van Ginkel et aI., 1992) for the

degradation of alkyl-trimethyl-ammonium compounds (not esterquats or quaternary

ammonium alcohols). In brief, the three alternative mechanisms that have been suggested

were 1) 00- and ~-oxidation of the alkyl chain resulting in betaine, which undergoes

progressive demethylation finally providing glycine; 2) Initial demethylation of the nitrogen

centre, then the splitting off of the nitrogen from the alkyl chain as ammonia and ~-oxidation

of the remaining alkyl chain; and 3) the splitting off of the alkyl chain from the quaternary

nitrogen atom with the release of the appropriate amine, the alkyl chain again undergoing ~

oxidation. However, from the results of this work no predictions can be made so far with

62


respect to the mechanisms responsible for the degradation of the QAAs and a careful

investigation is needed for the elucidation of the metabolic pathway(s) involved.

In the literature, most isolated bacteria able to degrade quaternary ammonium compounds

were found to be representatives of the genus Pseudomonas (Dean-Raymond & Alexander,

1977; Nishihara et al., 2000; Van Ginkel, 1996; Van Ginkel et al., 1992) and recently we

reported the isolation of a Pseudomonas strain growing with TM (Kaech & Egli, 2001). We

report here the isolation of microorganisms that are closer related at the phylogenetic level

(16S-rDNA sequence) to the genus of Zoogloea and Rhodobacter than to Pseudomonas.

Hence, the degradation of quaternary ammonium compounds seems not to be an exclusive

trait ofmembers of the genus Pseudomonas.

The properties of the isolated strains DM 1 and DM 2 were compared to those found in the

literature for the closely related genus Zoogloea. Their morphology (size, shape, flagellation

and storage of granules) and the inability to grow at pH 5 corresponded entirely with the

properties found for all species of Zoogloea as reported by Dworkin et al. (1999-2002).

However, only strain DM 1 exhibited movability of entire colonies on agar plates, cell

aggregation, sticking of aggregated cells to tubes and the lack of a visible zoogloeal matrix as

was described for the closest related strain Z ramigera Itzigsohn 1868AL (Dworkin et al.,

1999-2002; Friedman & Dugan, 1968; Friedman et al., 1969; Unz, 1971). The absence of

these properties for strain DM 2, however, does not imply that DM 2 is not a Zoogloea

species, since many exceptions with respect to cell aggregation have been reported (Dworkin

et aI., 1999-2002).

Since strain MM 1 showed 94 % identical bases only to the closest related strain Rhodobacter

sphaeroides based on the 16S-rDNA and did not exhibit the key properties found for all

species of the genus Rhodobacter, MM 1 most likely does not belong to the genus of

Rhodobacter, but must be considered to be a member of a new genus.

We have shown here that a consortia of microorganisms is not required for the degradation of

quaternary ammonium alcohols deriving from esterquat surfactants and that the pattern of

degradation for individual quaternary ammonium alcohols differs quite distinctly. Further

research will be focused on the enzymatic degradation pathways of TM, DM and MM to

elucidate the enzymatic mechanisms and specificity of the degradation of QAAs. This

information will be important with respect to the environmental behavior of these compounds

63

Chapter 3

and may help in designing readily and ultimately biodegradable alternative quaternary

ammonium compounds.

ACKNOWLEDGEMENTS

Dr. Ernst Wehrli, Laboratory for Electron Microscopy, ETHZ is gratefully acknowledged for

preparation of the electron micrographs of the isolated strains. Thanks also go to I.

Holderegger for CHNS-analysis and Christoph Werlen for 16S-rDNA sequencing of the

isolated strains.

64

Microbial oxidation of MM

4. Microbial oxidation of methyl-triethanol-ammonium

ABSTRACT

Methyl-triethanol-ammonium (MM) originates from the hydrolysis of the parent esterquat

surfactant, which is used as softener in fabric care. The initial conversion of MM was

investigated in cell-free extracts of the bacterial strain MM 1 able to grow with MM as sole

source of carbon, energy and nitrogen. Enzymatic activity transforming MM was located in

the particulate fraction of strain MM 1. The oxygen dependent reaction occurred also in the

presence of phenazine methosulfate as alternative electron acceptor. As soon as one ethanol

group of MM was oxidised to the aldehyde, a cyclic hemiacetal (and its stereoisomers) was

built by intramolecular cyclisation. The third ethanol group of MM was oxidised to the

aldehyde and the carboxylic acid sequentially. However, no further oxidation was observed

for the cyclic hemiacetal. The structurally related quaternary ammonium compounds

dimethyl-diethanol-ammonium (DM) and choline were oxidised in the particulate fraction of

strain MM 1 as well. Since DM contains two ethanol groups, only the cyclic product (and its

stereoisomer) was formed. With choline, the expected primary and secondary oxidation

products betainealdehyde and betaine have been detected. The observed oxidation of MM,

DM and choline was also present in the particulate fraction of strain MM 1 grown with

acetate or choline. The oxidation of MM, DM and choline is most likely catalised by the

same, constitutively expressed membrane-associated oxidoreductase.

65

Chapter 4

INTRODUCTION

The quaternary ammonium alcohols methyl-triethanol-ammonium (MM), dimethyl-diethanol

ammonium (DM) and 2,3-dihydroxypropyl-trimethyl-ammonium (TM) are the three mainly

used head groups in esterquat surfactants, which are applied as softeners in fabric care

(Krueger et al., 1998). The parent esterquat surfactants hydrolyse rapidly, abiotically and/or

biocatalysed, when reaching surface water or sewage treatment plants to the fatty acids and

the quaternary ammonium alcohols (QAAs) (Hellberg et al., 2000; Krueger et al., 1998;

Puchta et al., 1993; Simms et al., 1992). The biodegradability of both, the parent esterquat

surfactant and the quaternary ammonium alcohol, has been investigated in standard OECD

biodegradation tests. Based on these tests they are considered as readily and ultimately

biodegradable (Krueger et al., 1998; Puchta et al., 1993; Simms et al., 1992; Waters et al.,

2000). Whereas the fatty acids are expected to biodegrade via the common fatty acid

catabolism (~-oxidation), the enzymes involved in the degradation of the quaternary

ammonium alcohol MM (and the other QAAs) are not yet known. Considering the

widespread application of these QAAs and the development and design of similar

compounds, it is important to know the strategies and the pathways of their biodegradation.

Therefore, we set out to enrich and isolate microorganisms able to grow with MM and to

investigate the enzymatic mechanisms that are responsible for its breakdown. We isolated a

microbial strain growing with MM as sole source of carbon, energy and nitrogen (see

Chapter 3). In this work, the initial step in the catabolism of MM was investigated in the cell

free extracts of the isolated strain. The protein fractions were also examined with respect to

the ability to transform the structurally similar compounds TM, DM, and choline.


The quaternary ammonium alcohols (±)-2,3-dihydroxypropyl-trimethyl-ammonium (racemic

mixture), dimethyl-diethanol-ammonium and methyl-triethanol-ammonium were provided by

Unilever (SEAC Safety and Environmental Assessment Center, Unilever Research, Port

Sunlight, UK) as the iodine salts. All other compounds were purchased from Fluka, Buchs,

Switzerland, unless indicated otherwise.

66


Bacterial strains and cultivation. All experiments were performed with cells or cell-free

extracts of strain MM 1 isolated in our laboratory with MM as the sole source of carbon,

energy and nitrogen. An alignment of the 16S-rDNA sequence of strain MM 1 (EMBL:

AJ440751) to the sequences in the EMBL database (European Molecular Biology Laboratory,

Heidelberg, Germany) using the BLAST2 routine (Gish, 1996-1999) provided only 94 %

identities (best hit) of total 1434 base pairs to Rhodobacter sphaeroides (EMBL: X53854).

The physiological and morphological characterisation of strain MM 1 was reported previously

(Chapter 3).

Cultivation and storage of the strains was performed as described by Kaech & Egli (2001).

Preparation of cell-free extracts. Cells of strain MM 1 were grown in batch culture with 1 to

2 g r 1 of the indicated substrate. Cells were harvested in the late exponential growth phase by

centrifugation at 4 QC and 7000*g for 10 minutes (rotor: A 8.24, Kontron Instruments, Vietri

SuI Mare, Campania, Italy). Cells were washed once and resuspended after repeated

centrifugation with PB (50 mM). Before breakage of the cells - 20 mg r1 of DNAse I (EC

3.1.21.1, Aldrich, Milwaukee WI, USA) was added. Initially, 1,4-dithio-D,L-threitol (DTT,

2 mM) was added to the cell suspension as well, but later DTT was omitted, since it was

found to have no effect on enzyme activity in the protein fraction. The cells were broken by

two passages through a French press (Aminco, Urbana, USA) at 20000 psi. After each

passage the cell suspension and the French press were cooled with ice to 0 QC. The CE was

centrifuged for 30 min at 15000*g to remove unbroken cells and cell debris (rotor as above).

Subsequently, the cell-free extract was separated into a particulate (PF) and a supematant (SF)

fraction by ultra-centrifugation at 180000*g for one hour (rotor: TFT 65.13, Kontron

Instruments). The SF was removed and the pellet was suspended in PB (50 mM) and will be

referred to as the PF. Aliquots of the protein fractions were frozen at -20 QC until used in

assays. No reduction in activity was observed during freezing/thawing and storage. To

determine protein concentrations the Bio-Rad enzyme assay was used according to the

manual of the manufacturer (Bio-Rad Laboratories GmbH, Munich, Germany). BSA was

used as standard (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

67

Chapter 4

Enzyme activity assays. All assays were carried out in GC-Vials (Supelco Inc., Bellefonte

PA, USA) of 5 to 20 ml volume. Protein solutions were incubated at a pH of 7 and

thermostated at 30 QC under continuous stirring with a magnetic fly. The NMR samples were

prepared either by dilution with D20 (Aldrich, Milwaukee Wi, USA) or by carrying out the

assays with PB in D20. Protein concentrations used in the assays were always in the range of

0.4 and 1.0 mg mr I. To stop the enzymatic reaction, either aliquots removed from assays were

heated for 1 min in a water bath of 95 QC, or 1 M HCI was added (10 % of sample volume).

Before analysis, acidified samples were neutralised with 1 M NaOH (10 % of sample volume)

and the pH was controlled with test strips (Tritest pH 1 - 11, Macherey-Nagel, Dtiren,

Germany). IH NMR analysis indicated that using acid to stop the reaction did not change the

chemical composition of educts and products in samples. The oxygen dependence of the

reaction was monitored by carrying out experiments in sealed vessels with a continuous

nitrogen flow through the headspace. All solutions used in these experiments, except for the

protein stock solution, were bubbled with nitrogen for 10 min before adding them to the

assay. The electron acceptors NAD+, NADP+, PMS or INT were tested. NAD+, NADP+ and

PMS, final concentrations will be indicated at the appropriate place, were added to the assay

from stock solutions. INT was added to the reaction mixture from a saturated stock solution in

H20 (volume added to assays was 10 % of the total assay volume). For control experiments

protein solutions were heated in a water bath (- 95 QC) for 1 min prior to the addition of

substrate(s). Samples were stored at -20 QC prior to analysis. All enzyme specific activities are

given in nmol of converted substrate per min and per mg of protein (nmol min- I mg- I).

Chemical oxidation of MM. The oxidation of an ethanol group of MM to the corresponding

aldehyde was performed according to the method described by Dess & Martin (1983). BTF

was used as solvent (Ogawa & Curran, 1997) due to the poor solubility of MM in

dichloromethane. Saturated solutions of the oxidising reagent and MM in BTF were used for

the synthesis. After the reaction, residual MM and its oxidation products were extracted from

the BTF reaction mixture twice with H20. The combined water phase was freeze-dried and

the residual dissolved in D20 for NMR analysis.

68


Nuclear magnetic resonance spectroscopy. IH, 13C and 14N NMR spectroscopy were used

as analytical method to follow enzymatic activity and to elucidate the structure of the reaction

products. NMR spectra were recorded on a Bruker AMX-400 NMR spectrometer at 300 K

using a 5 mm broadband probe. The enzyme assays were directly performed in DzO buffer or

the samples spiked subsequently with DzO solution for the NMR lock signal. The NMR

experiments were performed without any further purification or filtration of the solutions. The

IH NMR (13C; 14N) spectra were recorded at 400.13 MHz (100.61; 28.9 MHz) with the

following parameters: 10.5 Ils (6.5; 13.5 Ils) 90° pulse lengths, appropriate number of

transients for reasonable SIN ratio, 8300 Hz (30300; 5800 Hz) spectral widths, 32k (64k; 16k)

data points, and 15 s (2.5, 1 s) relaxation delays. The I H NMR spectra were recorded with

presaturation of the water resonance using composite pulses (Bax, 1985). The 13C and 14N

NMR spectra were recorded using a proton decoupling field of 2.3 kHz during acquisition

(WALTZI6; Shaka et aI., 1983). The IH (13C) chemical shifts are given in parts per million

(ppm) relative to the signals of sodium 3-trimethylsilyl-tetradeutero-propionate (TSP) at

0.00 ppm (1.7 ppm).

The chemical shifts of the N-methyl groups of the substrates were used as internal reference

for the NMR spectra of the enzyme assays. The chemical shifts of 200 mM solutions of the

educts MM, DM, TM and choline in DzO were determined relative to TSP (18.7 mM) and are

given in Table 4.1.

The 14N chemical shifts of the same samples were determined relative to the signal of pure

nitromethane containing capillaries at 0.0 ppm and they were used as internal reference for the

NMR recorded during enzyme assays. The IH,14N HMQC (Bax et aI., 1983) experiments

were performed using the above mentioned 90° pulse lengths with the selection of a coupling

constant of 4 Hz showing the best results. The data was processed in the phase sensitive mode

to achieve better resolution in the 14N dimension for the methyl-nitrogen correlation signals.

IH,13C 2D correlation experiments were performed on a 5 mm broadband inverse probe with

z-gradient (100% gradient strength of 10 G cm-I) and 90° pulse lengths of 8.2 IlS eH) and

10.5 Ils (13C). The gradient selected HSQC (Davis et aI., 1992) (HMBC; Wilker et al., 1993)

experiments were performed with the selection of IH,13C coupling constants of 140 Hz

(5 Hz), gradient strengths of -40: 10 (15:9: 12), 2920 x 4800 Hz spectral widths with a carbon

decoupling field of 3.7 kHz for the HSQC experiments (GARP decoupling; Shaka et aI.,

69

Chapter 4

1985). The data matrices of 1024 x 256 were zero filled to 1024 x 1024. The HSQC-TOCSY

(Palmer et al., 1991) spectra were recorded with the selection of I JeH,13C) = 140 Hz and a

29 f.ls 90° pulse length for the TOCSY transfer with a total mixing time of 114 ms, applying

the above mentioned carbon decoupling conditions, data matrices and processing conditions.

The NOESY (Jeener et a!., 1979) spectra were recorded with data matrices of 1024 x 256

(spectral widths of 2400 x 2400 Hz) with presaturation of the water resonance during the

relaxation delay (2 s) and the mixing time (800 ms).

Quantitative determinations were performed using the IH NMR signals of the CH3-groups of

the corresponding compounds. The sum of all CH3-group integrals of a spectrum was set to

100 % and the amount of the educts and products were calculated according to their relative

intensities.

Table 4.1. I R, l3C and 14N chemical shifts of MM, DM, TM and choline (200 mM) in DzO in ppm and

coupling constants J( l3C,14N) in Rz.

Position MM DM Choline TMN-CH3 OeH) 3.26 3.23 3.21 3.24

O(13q 54.6 56.7 58.4 58.7IJ(13C,14N) 3.7 3.8 3.9 3.9

N-CH2 OeH) 3.68 3.60 3.52 3.43/3.48

oe3q 69.0 70.8 72.0 72.7IJ(13C,14N) 2.6 2.9 3.1 3.2

CH2-OH OeH) 4.07 4.07 4.07 3.60O(13q 59.7 59.9 60.1 70.0

CH-OH OeH) 4.30O(13q 68.2

Oe4N) -322.5 -328.1 -333.8 -334.1

Cation chromatography. Disappearance of MM in enzyme assays was also followed by

cation exchange chromatography using the following equipment: IonPac CTC-1 cation trap

column, IonPac C012 guard column, analytical IonPac CS12 4 mm column, CSRS-ULTRA

4 mm suppressor (external mode with tetrabutylammoniumhydroxyde 50 mM), and CD20

conductivity detector (Dionex, Olten, Switzerland). Elution was performed with HCI 15 mM

at a flow rate of 1 ml min- I. MM eluted 10 minutes (peak width - 2 min) after sample

injection.

70


Oxygen uptake measurements. Oxygen consumption was measured with a Clark-type

oxygen electrode (Rank Brothers, Cambridge, UK). Respiration was recorded before and after

the addition of MM to 1 ml of the protein solution in PB (0.4 - 1.0 mg protein mrl of PF) in a

closed reaction vessel at 30°C. To monitor the effect of KCN on Oz consumption, KCN was

added to a final concentration of 5 mM to a running assay with MM after a running time of 5

minutes. To detect possible HzOz production, catalase (EC 1.11.1.6, from bovine liver, Fluka,

Buchs, Switzerland) was added to an assay with MM after running for 5 minutes. The

presence of catalase in the PF was tested by adding HzOz to the protein mixture in the oxygen

electrode (without the substrate MM).

RESULTS

Enzymatic consumption of MM in cell-free extracts of strain MM 1. In enzyme assays,

disappearance of the quaternary ammonium alcohol MM was observed in cell-free extract

(CFE) and in the particulate fraction (PF) of MM 1 cells grown with MM as sole source of

carbon, energy and nitrogen (Figure 4.1). No consumption of MM was found in enzyme

assays performed with the soluble fraction (SF) and in control experiments with inactivated

extracts (CFE, SF and PF). Thus, the observed disappearance of MM must be mediated by

membrane-associated enzymes. Since the lH NMR spectra of reaction mixtures with the CFE

and the PF showed identical behaviour (disappearance of substrate and formation of the same

products), only the PF was used for further studies of the initial degradation step.

Simple stirring of the reaction mixture in open vials was sufficient to maintain MM

consuming enzyme activity and no additional reaction partners were required. The addition of

NAD+ and NADP+ to a final concentration of 2 mM did not enhance this activity. However,

under nitrogen atmosphere only a background activity for MM was detected (Figure 4.2,

initial slopes). This suggests an oxidative reaction to be responsible for the conversion of

MM. Oxygen consumption was confirmed and followed in a Rank oxygen electrode.

Immediate interruption of oxygen uptake was observed by addition of KCN to a running

assay. No HzOz was produced during the reaction as often found for oxidases (transferring the

electrons directly to oxygen), since the addition of catalase to an assay did not lead to oxygen

production. Moreover, no catalase activity was present in the PF, since addition of HzOz to the

71

Chapter 4

PF did not result in oxygen production either. These findings suggest that the electrons are

transported via the respiratory chain present in the particulate fraction. However, this is no

final proof, since cyanide might not only have interacted with terminal oxidases but also with

the MM-consuming enzyme.

As alternatives to oxygen the electron acceptors PMS, NAD+ and INT were tested under

anaerobic conditions. If the transport of the electrons to the terminal electron acceptor

occurred via the respiration chain one would also expect INT to be able to substitute for

oxygen under anaerobic conditions, since INT is known to act as an acceptor for electrons

deriving from the respiration chain. PMS, on the other hand, is known to act as a redox

mediator for many redox enzymes for which the natural redox mediating compounds are not

known. After incubation of enzyme assays for 3 h under N2 (di-nitrogen), the electron

acceptors were added to the reaction mixture and the change in MM concentration was

followed by IH NMR spectroscopy (Figure 4.2). PMS was the only reactant acting as

alternative electron acceptor. Although INT was not used as electron acceptor, this does not

exclude that the electrons are transported via the respiration chain.

1401201008060

........."'0... _

-0-- 0 0- --- ---0

4020

1.2

1.0

0.8~

.2l 0.6:2:2

0.4

0.2

0.00

Time [min]

Figure 4.1. Disappearance of MM (0) in an enzyme assay with particulate fraction of strain MM 1

grown on MM as sole source of carbon, energy and nitrogen, as determined by cation

chromatography. Activity of the particulate fraction based on the initial slope (solid line) was

260 nmol min- l mi l. The outlier indicated in brackets was not used to determine the initial slope.

72


Up to an initial MM concentration of 2 g r l, the substrate was completely consumed and

transformed into products within 2 h of incubation. Increasing the initial MM concentration to

6 g r l and up to 15 g r l, the amount of consumed MM decreased to 82 % and 12 %,

respectively. lH NMR data of an assay with 6 g r l (Figure 4.3) resulted in a specific rate

(initial slope) for MM disappearance of 324 nmol min- l mi l and from the cation

chromatography data of an assay with 1 g r l of MM an initial slope of 260 nmol min- l mg- l

can be estimated (Figure 4.1).

0.6 -r---------------------,

6543Time [h]

2

0.0 +---,----,----r------,r----.---lo

0.4

0.1

0.2

,g 0.3::2:::2:

Figure 4.2. Consumption of MM (0.5 g r l) in the particulate fraction of strain MM 1 (0.43 mg ml- I of

total protein) under anaerobic conditions as determined by IH NMR spectroscopy. After three hours,

the electron acceptors (0) PMS (4.5 mM), (I::::.) NAD+ (3.2 mM), or (D) INT (0.5 ml from a saturated

stock solution in water to 4.5 ml of the reaction mixture) were added to independent enzyme assays

(arrow). Only PMS stimulated activity significantly. A background level of specific enzyme activity of

approximately 34 nmol min- I mg- I was always detected in experiments under anaerobic conditions

(indicated by the initial slopes).

73

Chapter 4

Substrate specificity. The structurally related compounds 2,3-dihydroxypropyl-trimethyl

ammonium (TM), dimethyl-diethanol-ammonium (DM) and choline were tested in the

particulate fraction of MM 1 (cells grown with MM) under the same experimental conditions

as those used for MM. Whereas the PF exhibited activity with DM and choline as well, no

consumption of TM was observed. However, the specific activity with DM

(48 nmol min- I mg-I) and choline (23 nmol min- I mg-I) was much lower than with MM

(324 nmol min- I mg-I). Figure 4.3 shows enzyme assays with MM, DM and choline with

initial concentrations of 6 g r l and a protein concentration of 0.4 mg mr l. As for MM, the

activities with DM and choline were 02-dependent, only a slight residual activity was found

under di-nitrogen atmosphere, and PMS also acted as alternative electron acceptor. It should

be pointed out that although strain MM 1 was unable to grow with DM, this QAA was

transformed in the PF of MM-growing cells.

7

6

- 5.9(J)c

4(5.!:U

~ 30

~ 2~

----------------1)

[0]... ... ... ..., , , ,

"0.............0.

--- .... _------------'0

1084 6Time [h]

2

O+----,.------,,....-:l<.-----..----r-------!

o

Figure 4.3. Substrate specificity of the particulate fraction of strain MM 1 grown with MM. The

disappearance of (0) choline, (6) DM, and (D) MM (control) was followed by lH NMR

spectroscopy. Initial substrate concentrations of 6 g r1 and a protein concentration of 0.4 mg ml-1 were

used in all assays. Solid lines indicate initial slopes. Initial specific activities were 23 nmol rnin-1 mg-1

for choline, 48 nmol rnin-1 mg-1 for DM, and 324 nmol rnin-1 mg-1 for MM. The outlier is indicated in

brackets.

74


Products from choline. The lH NMR spectra of assays using the particulate fraction of strain

MM 1 and choline as substrate provided two product signals of methyl groups at 3.23 and

3.28 ppm additionally to the educt signal (Figure 4.4a). These products were identified as the

primary and secondary oxidation products betainealdehyde (hydrated form) and betaine,

respectively. The assignment of the NMR signals to the structures postulated was performed

based on the JH,l3C HSQC (correlation over one atom bond) and the lH, l3C HMBC spectra

(correlation over 2 or 3 atom bonds). All observed 2D correlation signals confirmed the

proposed structures. The chemical shifts 8eH) and 8(l3C) of both compounds are given in

Table 4.2 and the structures are shown in Figure 4.5a. The l4N NMR signal of

betainealdehyde was detected as broad shoulder of the main signal betaine. The definite

assignment of the signals was performed using a lH,l4N HMQC correlation. Whereas the l4N_

signal of choline was very narrow (~VIl2 =0.5 Hz), the signals of the products showed a clear

broadening of the lines (4.0 - 4.3 Hz).

Products from DM. The consumption of DM in particulate fraction enzyme assays was

monitored by lH NMR. The spectral region of the N-methyl resonances is shown in Figure

4.4b together with the postulated structure of the formed hemiacetal. This compound exists as

two enantiomers and only the structure of the 2R configuration is shown. Two lH NMR

signals of the diastereotopic methyl groups at 3.26 and 3.35 ppm were observed. All detected

lH, l3C correlation signals (HSQC and HMBC) confirmed the postulated structure

(Figure 4.4b). Chemical shift assignments are given in Table 4.3. The correlations H-(2)/C

(3), H-(5)/C-(1,4,6) and H-(6)/C-(1,4,5) observed in the HMBC spectrum demonstrated the

connectivity over the heteroatoms Nand O. With the lH,lH NOESY spectrum, the

neighbourhood of the anomeric proton H-(2) with the methyl group H-(6) was confirmed. A

8e4N) of -334.3 ppm was determined, corresponding to a deshielding of 6.2 ppm compared to

the educt signal. The line width of the product resonance (1.5 Hz) was only slightly enhanced

compared to the educt signal (0.5 Hz).

75

Chapter 4

Choline

Betainealdehyde

(a)

(b)

D

I I I I I8(1H) 3.40 3.35 3.30 3.25 3.20 ppm

Figure 4.4. lH NMR spectral region with signal assignements of the methyl groups of (a) choline and

(b) DM and their oxidation products. Initial substrate concentrations of 6 g r l, a protein concentration

of 0.5 mg ml- l and an incubation time of 12 hours were used for the enzyme assays.

Table 4.2. lH, 13C and l4N (with line width, ~Vl/2) chemical shifts of the products betaine and

betainealdehyde (hydrated form) obtained by degradation of choline in the particulate fraction of strain

MM 1. Chemical structures are shown in Figure 4.5a.

Betaine

Betainealdehyde

Position BeH) B(13e)[ppm] [ppm]

N-CH3 3.28 58.1N-CH2 4.02 69.9COOH 173.0N-CH3 3.23 58.9N-CH2 3.43 73.1CH-(OHh 5.56 89.6

Be4N) Llv 1/2

[ppm] [Hz]

-334.9 4.3

-335.2 ca.4

Table 4.3. lH and 13C chemical shifts of the product obtained from the degradation of DM in the

particulate fraction of strain MM 1. The positions of the carbon atoms in the molecule are shown in

Figure 4.5b.

Position of C NumberofH BeH) B(l3e)[ppm] [ppm]

1 2 3.31/3.56 67.32 1 5.40 92.43 2 4.03/4.34 60.64 2 3.52/3.52 64.75 3 3.35 58.76 3 3.26 57.6

76


(a) Structures of choline and its oxidation products

OH

IJOH IJ-OH

/N\ /N\

Choline Betainealdehyde(hydrated form)

Betaine

(b) Structures of DM and its oxidation product

DM

r 2 OH

N+""'''~s/~O

4

Oxidation product

(c) Structures of MM and its oxidation products

MMHOI+~OH

/N\/OH

7Ri6 2N+",,·,,~OH

S/ "y/304

trans-Products

IS 2

~ N+."",,~OHV "y/30

6 4

eis-Products

trans-/eis-1 :

trans-/eis-2:trans-/eis-3:

R = CH20H

R = CH(OH)2R=COOH

Figure 4.5. Chemical structures ofthe substrates (a) choline, (b) DM, (c) MM and their corresponding

oxidation products. Numbers indicate the position of carbon atoms.

77

Chapter 4

Table 4.4. IH and 13C chemical shifts of the products found by degradation of MM in the particulate

fraction of strain MM 1. The positions of the carbon atoms in the molecules are shown in Figure 4.5c.

Primary products: trans-l cis-l

Position of C NumberofH 8ctH) 8(13C) 8(IH) 8(13C)(ppm) (ppm) (ppm) (ppm)

1 2 3.39/3.69 66.6 3.39/3.61 66.7

2 5.44 92.4 5.41 92.3

3 2 4.05/4.40 60.0 4.09/4.31 61.0

4 2 3.57/3.69 64.3 3.59/3.65 64.2

5 3 3.42 55.5 3.32 54.1

6 2 3.65 71.2 3.78 72.1

7 2 4.12 59.4 4.12 59.6

Second. products: trans-2 cis-2Position of C NumberofH 8ctH) 8(13C) 8ctH) 8(l3C)

(ppm) (ppm) (ppm) (ppm)

1 2 * * * *

2 1 * * 5.40 *

3 2 * * * *

4 2 3.58/3.74 64.8 3.61/3.69 64.7

5 3 3.46 55.8 3.36 54.6

6 2 * * * *

7 1 5.64 89.1 5.66 89.3

Tert. products: trans-3 cis-3

Position of C NumberofH 8ctH) 8(13C) 8('H) 8(13C)(ppm) (ppm) (ppm) (ppm)

1 2 3.50/3.85 66.0 3.66/* 65.8

2 1 5.42 92.5 5.40 92.5

3 2 4.03 /4.40 60.1 4.09/4.32 60.8

4 2 3.61/3.87 63.7 3.66/3.76 63.9

5 3 3.52 55.5 3.41 54.6

6 2 4.02/* 69.7 4.09/4.24 70.0

7 172.9 173.4

* not assignable

78


Products from MM. The degradation of MM in PF enzyme assays (2 or 6 g r 1 MM)

provided at first an unidentifiable mixture of products. In the IH NMR spectrum, at least 7

different resonances of nitrogen-bound methyl groups were found in addition to the signal of

the educt MM (Figure 4.6b). As the assay proceeded, the signal of the methyl group of MM

(3.26 ppm) disappeared and the methyl resonances of the primary products at 3.32 and

3.42 ppm increased. With time, the primary product signals (assigned as trans-/eis-l in Figure

4.6) declined again while several secondary product peaks increased (Figure 4.6c). To

simplify the analysis of the spectra, MM was oxidised to the aldehyde by the Dess-Martin

method, since an oxidation was expected from the results obtained in anaerobic assays and

02-uptake experiments. The relevant section of a IH NMR spectrum of the synthesised

product, showing the methyl resonances, is depicted in Figure 4.6a. Additionally to the educt

signal (methyl groups of MM) two more singlets were detected at 3.32 and 3.42 ppm. They

correspond to the first evolving signals from the enzyme assay. Based on the IH NMR

spectrum two primary, diastereomeric oxidation products (trans-l and eis-I) were postulated

(Figure 4.5c). The stereochemical relations eis or trans are defined by the relative

configuration of the substituted carbon C-(6) to the OH group at the anomeric carbon C-(2).

As soon as one ethanol group of MM was oxidised an intramolecular reaction with a second

ethanol group lead to the cyclic hemiacetal (6-ring), similar to the cyclic structures found in

sugars (for glucose more than 99 % is usually present in the hemiacetal form; Koolman &

Rohm, 1998). Both diastereomeric products evolved in equal amounts.

In the l3C NMR spectrum 14 additional signals to the educt signals were detected. The IH, l3C

correlation over one bond (HSQC) showed distinct cross signals for 11 of these signals. Due

to extensive overlaps of the signals at about 4.1 ppm eH) and 59.8 ppm (l3c) with the strong

correlation signals of the residual educt, no unequivocal assignment was possible. Definite

assignment of the IH and l3C chemical shifts of trans-l (H-(3, 4, 7), C-(3, 7); Figure 4.5c) and

eis-l (H-(3, 7), C-(7); Figure 4.5c) was achieved by performing a HSQC-TOCSY experiment.

The IH, l3C HMBC spectrum showed all necessary correlation signals across the hetero atoms

Nand 0, which were essential to confirm the product structures. Moreover, the correlation

between the protons H-(2) and H-(5) (Figure 4.5c) found in the IH,lH NOESY spectrum

confirmed the relative configuration of eis-I. The corresponding protons of trans-l did not

show any cross signal.

79

Chapter 4

MM~ trans-/cis-1

811 trans-/cis-2

~ trans-/cis-3

(a)

?

?

(c)

(d)-A.. ~ -A.j

(e)

I3.50

I3.45

I3.40

I3.35

I3.30

I3.25 ppm

Figure 4.6. I H NMR spectra with assignments of the methyl groups of MM and its oxidation products

(a) from chemical oxidation of MM, (b) from an enzyme assay using the particulate fraction of MM

grown cells of strain MM 1 with 6 g r l of MM, 0.5 mg ml- I of protein, 12 hours of incubation, (c)

from a similar enzyme assay with 2 g r l of MM, 0.5 mg ml- I of protein and 5 hours of incubation, (d)

from the culture liquid of strain MM 1 grown in batch culture with an initial concentration of

350 mg r l of MM after 7 hours of incubation (exponential phase) and, (e) from the same culture after

12 hours (stationary phase) of incubation.

80


The 14N NMR signals were assigned via IH,14N HMQC correlation of the methyl protons at

3.32 and 3.42 ppm with the 14N resonances of both diastereomers at -329.0 ppm (trans-l) and

-327.8 ppm (eis-I) with line widths !1V1l2 of 2.5 and 2.3 Hz, respectively (Figure 4.7). The

average of 8ct4N) was -328.4 ppm and showed a shielding of 5.9 ppm relative to the 14N

signal of the educt. The chemical shifts of the primary oxidation products trans-l and cis-l

are listed in Table 4.4.

trans-2

~ ~trans-3

trans-1 G

-330.0

-329.0

Figure 4.7. IH,14N HMQC spectrum with assignment of signals to the oxidation products trans-/cis-l,

2 and 3 from an enzyme assay using the particulate fraction of MM-grown cells of strain MM 1. Initial

concentration of MM: 2 g r 1; protein concentration: 0.5 mg ml-1

; incubation time: 5 hours. MM was

completely converted to products under these conditions.

81

Chapter 4

In the IH NMR spectrum of the enzyme assay performed with MM at least 5 additional

signals with significant intensities were visible (Figure 4.6b,c), which belonged to the N

methyl groups of other products. In the IH,l3C-HMBC spectrum (correlation of the chemical

shifts over 2 - 3 atom bonds) explicit cross signals of protons in the region of 4.0 - 4.2 ppm

with carbon atoms at 172.9 and 173.4 ppm were detected. Each of these proton signals

correlated with three further carbon atoms providing very similar chemical shifts as found for

the products trans-I and eis-I. Based on these findings, two tertiary oxidation products trans

3 and eis-3 (Figure 4.5c) were postulated with the IH resonances of the methyl groups at 3.41

and 3.52 ppm (Figure 4.6b,c). The two compounds are cyclic hemiacetals as described for the

primary products with the third ethanol group oxidised twice to the corresponding carboxylic

acid. The chemical shifts 8eH) and 8(l3C) of both cis- and trans-3 are given in Table 4.4.

Again, both products were found in equimolar amounts.

The IH, l3C HMBC correlation of the methyl protons in position 5 with the carbon atoms 1,4

and 6 (Figure 4.5c) as well as the correlations found with the IH,l3C HSQC spectrum

confirmed the structural elements linked to the nitrogen atoms. The IH, l3C HSQC-TOCSY

experiment provided the assignment of the IH and l3C chemical shifts at positions 2 and 3

(Table 4.4). The IH,lH NOESY spectrum showed unequivocally that the signal of the methyl

group at 3.41 ppm correlated with H-(2), therefore confirming the structure eis-3. The

structure of the trans-3 product was confirmed via the correlation between the protons H-(6)

and H-(2) (Figure 4.5c). In the ID 14N NMR spectra the signals of the different products were

not resolved clearly due to strong overlapping, and in samples with low product

concentrations these signals were hardly detectable. Therefore, the assignment of the signals

to structures trans-3 and eis-3 was performed via the IH,14N HMQC correlation (8(14N):

trans-3: -330.5 ppm; eis-3: -329.9 ppm; Figure 4.7).

Additionally to the previously assigned IH NMR signals, two further methyl singlets were

detected at 3.36 and 3.46 ppm (Figure 4.6b,c), which were supposed to belong to the cyclic

products trans-2 and eis-2 (hydrate form of the aldehyde, Figure 4.5c). Because products with

a higher oxidation state (carboxylic acid) were found and confirmed, one would also expect

these intermediates.

Since most resonances of the IH and l3C signals probably lay beyond the signals trans-/cis-I,

trans-/eis-3 and MM (Figure 4.6b,c), no final prove of eis-2 and trans-2 was possible without

82


purification of individual products from the mixture. However, several indications were found

for their existence and the chemical shifts 8eH) and 8(13C) giving evidence to the proposed

trans-2 and eis-2 structures are listed in Table 4.4.

The IH,13C HMBC correlations of the methyl protons H-(5) (Figure 4.5c) provided cross

signals to the carbon atoms at 64.7 and 64.8 ppm (probably C-(4)). The correlation signals to

C-(I) and C-(6), supporting these structures were not detected unequivocally. Additional hints

for the expected structures were observed in the IH,lH NOESY spectrum: trans-2 showed a

correlation signal of H-(5) with H-(7), whereas for cis-2 a correlation of H-(5) with H-(7) as

well as with H-(2) was detected. Based on this steric interaction the relative configuration of

cis-2 at the nitrogen atom was deduced. The corresponding 14N chemical shifts of -330.3 ppm

(trans-2) and -329.7 ppm (eis-2) were observed (Figure 4.7).

All coincident pairs of the described diastereomers showed a stronger shielding of the

nitrogen nucleus in the eis-configuration compared to the isomers trans. It was not possible to

identify the structure of the product with its methyl singlet at 3.34 ppm (Figure 4.6b,c).

During batch growth of strain MM 1 the substrate MM continuously decreased in the

exponential phase until it had completely disappeared when reaching the stationary phase

after about 11 hours (Chapter 3). However, at the end of such batch cultures, high residual

concentrations of carbon and nitrogen were left in the culture broth. Therefore, supematants

of batch cultures in the exponential and stationary phase were investigated by IH NMR

spectroscopy with respect to the presence of excreted metabolites. Such products were found

in considerable amounts in the culture liquid and the majority of them were identified as

tertiary oxidation products eis-/trans-3 (Figure 4.6d and e). Based on the measured residual

carbon and organic nitrogen concentrations (Chapter 3) these products accumulated during the

exponential growth phase to a maximum final concentration of 30 mole-% of the initially

provided MM. In the exponential phase, transient accumulation of low concentrations of the

primary oxidation products cis-/trans-l (Figure 4.6d) were detected as well. However, they

disappeared again in the late exponential phase (Figure 4.6e).

83

Chapter 4

Expression of enzymes in the particulate fraction of MM 1. The expression of the enzymes

responsible for the degradation of MM, DM or choline was studied with the help of enzyme

assays employing the particulate fraction of MM 1 cells grown either with MM (control),

choline or acetate. Acetate was chosen as a substrate because it does not contain nitrogen and

is catabolised via a different metabolic pathway compared to that known for choline. For the

growth with acetate N~Cl was used as nitrogen source. The experiments were performed

with 2 g r l of the carbon substrate and a protein concentration of 0.5 mg mr l of the protein

fraction. After 5 hours of incubation the reaction was stopped and the assays were analysed by

I H NMR spectroscopy. In all assays transformation of MM, DM and choline to the

corresponding products was observed as described above. MM always was converted to the

highest extent, followed by DM and choline (Figure 4.8).

Strain MM 1 grown with:

Choline

100 ~,="....---_......!-.,--!----~.,--!------L....,

~ 80E::JC/)c8 60Q)

co....en 40.0::JC/)

<5~ 20o

oMM DM Ch MM DM Ch MMDM Ch

Substrates tested in enzyme assays

Figure 4.8. Substrate specificitiy of the particulate fraction of strain MM 1 grown with different

substrates. The bars indicate the percentage of consumed substrate after 5 hours of incubation (single

experiments). The enzyme assays were performed with initial concentrations of MM, DM and choline

(Ch) of 2 g rI, each, and protein concentrations of 0.5 g rl.

84


DISCUSSION

All detected enzyme activities were associated with the membrane of strain MM 1 making

purification of the enzymes difficult or even impossible. Therefore, enzyme assays for the

investigations of the catabolic pathways were performed with the particulate fraction of strain

MM 1.

The responsible enzyme for the initial degradation of methyl-triethanol-ammonium (MM)

most probably belongs to the membrane-associated oxidoreductase group of enzymes. Under

aerobic conditions, molecular oxygen acted as electron acceptor. Based on the performed

experiments, the electrons derived from MM were transported to molecular oxygen rather via

the respiration chain present in the particulate fraction than directly by the "MM

oxidoreductase". However, the unequivocal elucidation of the path of the electrons requires

additional investigations.

The proposed degradation pathway of MM is depicted in Figure 4.9. The products trans-lcis-l

and trans-lcis-2 are expected to undergo further degradation. Obviously, the continuing

degradation of the ring structure is not mediated by the "MM-oxidoreductase" since the cyclic

hemiacetals prevented further oxidation of the ethanol and acetaldehyde groups involved in

the cycle. The products trans-Icis-3, with the third ethanol group oxidised to the carboxylic

acids, are most likely dead-end metabolites, because considerable amounts of these

compounds were released into the culture broth and remained untouched in batch cultures of

strain MM 1. Consequently, one has to take into account that these metabolites may

accumulate in the environment. To investigate the possible presence of these compounds in

the environment, extensive studies in complex environmental systems, i. e. river water or

sewage treatment sludge would be required turning one's attention to the analysis of the

described metabolites. Detection of such metabolites in the environment would greatly

influence the design of new similar compounds.

The cyclic oxidation products (trans-Icis-l, 2 and 3) of MM each existed as 1: 1 mixture of the

two diastereomers. This fact may be interpreted in two ways: Either the enzyme mediating the

first oxidation step to the cyclic hemiacetals trans-Icis-l was not a diastereoselective reaction

(as would be expected) and/or this cyclic hemiacetal underwent a rearrangement with the

remaining ethanol group in the aqueous environment.

85

Chapter 4

Regarding the substrate specificity of the particulate fraction, the oxidation of DM and

choline proceeded under the same conditions as that found for MM, whereas TM remained

untouched. Additionally, the MM-, DM- and choline-consuming activity was independent on

the growth substrate used for strain MM 1. This suggests that one and the same constitutively

expressed enzyme catalyses these reactions. Since strain MM 1 was able to grow with choline

as well and the ability to oxidise choline is widespread amongst microorganisms (Kortstee,

1970) the enzyme described even may be a choline oxidoreductase with a broad substrate

specificity. Based on these findings, the following conclusions can be drawn with respect to

the described oxidoreductase. Free ethanol groups, as present in MM, DM and choline, are

essential to undergo oxidation in the active enzyme and/or the 2,3-dihydroxy-propyl part of

MM

trans-/cis-1

trans-/cis-2

~2e-+2H'OH

\,••/\OH ~/V-

0

~

H20-.,J Il\.... Further breakdownOH 20-+2/----------

HO~ OHN+''''''''.!\

/V- 0

~2e-+2H'trans-/cis-3

o

HO~ OH

N+''''''''.!\/V- 0

Excretion asdead-end metabolite

Figure 4.9. Proposed pathway for the initial degradation steps of MM in strain MM 1. Only trans

structures with the absolute configuration 2R, NS are shown.

86


TM prevented the molecule to fit into the active site. Obviously, the presence of a quaternary

nitrogen was not sufficient to allow enzymatic oxidation of the hydroxyl groups. Since DM

was oxidised in the particulate fraction but did not serve as a growth substrate, strain MM 1

was probably hampered in the transport of this compound into the cell.

Membrane-associated oxidoreductases consuming choline with similar properties as described

here were also detected in Pseudomonas aeruginosa and Escherichia coli (Bater & Venables,

1977; Lamark et al., 1991; Nagasawa et al., 1976; Russell & Scopes, 1994). Surprisingly,

these oxidoreductases (choline dehydrogenases) specifically oxidised choline to

betainealdehyde only, without further oxidation to betaine. Enzymes mediating both oxidation

steps were characterised by Ohta-Fukuyama et al. (1980) and Ikuta et al. (1977) from

Alcaligenes spec. and Arthrobacter globiformis, respectively. However, these enzymes were

soluble oxidases and both of them produced HzOz.

The particulate fraction described in this work exhibited a specific acitivity with choline in the

range of about 25 nmol min- I mg- I. This corresponds well to the specific activity range for

choline dehydrogenases in cell-free extracts and particulate fractions of

7 -78 nmol min- I mg- I reported in the literature (Boncompagni et al., 1999; Nagasawa et al.,

1976; Pocard et al., 1997).

The presented study suggests that the oxidation of MM might be linked to the oxidation of

choline. This raises the question, whether or not the degradation of quaternary ammonium

alcohols in general is related to the choline degradation pathway or whether different

strategies are responsible for the degradation of different quaternary ammonium alcohols. To

answer this question further investigations at the enzyme level using different microorganisms

able to grow with TM and DM are needed.

87

eite Leer /Blank leaf

Microbial degradation of TM

5. Microbial degradation of 2,3-dihydroxypropyl-trimethyl-ammonium

ABSTRACT

2,3-dihydroxypropyl-trimethyl-ammonium (TM) originates from the hydrolysis of the parent

esterquat surfactant, which is widely used as softener in fabric care. Judged on OECD

standard biodegradation tests simulating complex biological systems, TM is supposed to be

biologically degraded to biomass and CO2 when reaching the environment. However, the

degradation mechanisms of TM have not been elucidated so far. The initial step of breakdown

of TM was investigated in cell-free extracts of strain P. putida TM 1, a bacterium able to

grow with TM as sole source of carbon, energy and nitrogen and to degrade this compound to

completion. TM-consuming activity was located in the particulate fraction of P. putida TM 1.

Trimethylamine was split from TM without the addition of any cofactors and independent of

the presence of oxygen. Therefore, the responsible enzyme was supposed to be a membrane

associated lyase. Unfortunately, the structures of the remaining products derived from the

propyl moiety have not been identified so far, although their presence was detected by

different analytical methods. "TM-Iyase" appears to be an inducible enzyme because the

ability to catabolise TM was solely observed in the particulate fraction of TM-grown cells and

no activity was found in the particulate fraction of cells grown with acetate or choline. Except

for choline, none of the tested structurally related compounds such as dimethyl-diethanol

ammonium, methyl-triethanol-ammonium (both also used as head groups in esterquat

surfactants), betaine, D- or L-carnitine, were converted in the particulate fraction. However,

choline was transformed by a different mechanism, namely by an oxygen-dependent reaction.

The products from choline were identified as the primary and secondary oxidation products

betainealdehyde and betaine. All this indicates that the initial TM degradation is a highly

specific reaction and that there is no involvement of enzymes responsible for choline

catabolism.

89

Chapter 5

INTRODUCTION

The quaternary ammonium alcohol 2,3-dihydroxypropyl-trimethyl-ammonium (TM),

dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) belong to the

three mainly used head groups in cationic fabric softeners (Krueger et al., 1998). The parent

esterquat surfactants consist of the quaternary ammonium alcohols (QAAs) esterified at two

alcohol groups with long chain fatty acids deriving from tallow. Since the esterquat

surfactants are produced in considerable amounts worldwide (probably more than 100000

tons; Krueger et al., 1998), they have been investigated extensively in DECD test procedures

and by monitoring their environmental concentrations. When reaching surface water or

sewage treatment plants they hydrolyse rapidly, abiotically and/or biocatalysed, to the fatty

acids and the corresponding QAAs. Based on DECD biodegradation tests, both, the parent

compounds as well as their products are judged as readily and completely biodegradable

(Giolando et al., 1995; Krueger et al., 1998; Matthijs et al., 1995; Puchta et al., 1993; Simms

et al., 1992; Waters et al., 1991; Waters et al., 2000). Whereas the fatty acids are expected to

degrade via the common fatty acid metabolism (l3-oxidation), the degradation mechanisms for

the QAAs are not known to date. Details are limited to some DECD die-away tests, which

provided different degradation rates and patterns for the three QAAs (Hales, 1998).

Considering the large quantities used worldwide and the development of new similar head

groups with improved environmental properties, it is important to elucidate the strategies and

mechanisms involved in their biodegradation. Therefore, we isolated microorganisms able to

grow with these QAAs as a basis for further biochemical investigations.

Using the quaternary ammonium alcohol TM as a sole source of carbon, energy and nitrogen,

a Pseudomonas putida strain, referred to as P. putida TM 1, was isolated and described in

detail by Kaech & Egli (2001). In this study, the initial degradation mechanism of TM in cell

free extract of this isolate was investigated.


Chemicals. The quaternary ammonium alcohols (QAAs) (±)-2,3-dihydroxypropyl-trimethyl

ammonium (TM, racemic mixture 1:1), dimethyl-diethanol-ammonium (DM) and methyl-

90


triethanol-ammonium (MM) were provided by Unilever (SEAC Safety and Environmental

Assessment Center, Unilever Research, Port Sunlight, UK) as the iodine salts. 14C-labelled

TM was supplied as stock solutions in methanol. The activity of 1 /11 of 14C-methyl-labelled

TM and 14C-propyl-labelled TM (label at propyl carbon in position 3) was 36000 dpm and

85000 dpm, respectively. All other chemicals were purchased from Fluka, Buchs,

Switzerland, unless indicated otherwise.

Bacterial strains and storage. Experiments were performed with cells and cell-free extracts

of strain P. putida TM 1, which was isolated with TM as the sole source of carbon, energy

and nitrogen (Kaech & Egli, 2001). In some experiments, also the particulate fraction of

isolate DM 2 was used, a strain isolated originally with DM, but also able to grow with TM

and using both substrates as the sole source of carbon, energy and nitrogen. The 16S-rDNA

sequence of isolate DM 2 indicated identities to Zoogloea ramigera Itzigsohn 1868AL of 98 %

(best hit, total 1448 base pairs, Chapter 3). For short-term storage all strains were plated on

lO-fold diluted tryptic soy agar or on agar plates containing SM and a selective carbon source.

For long-term preservation all strains were suspended in 30 % glycerol and stored at -80 QC.

Cultivation of microorganisms. Cultivation of the microorganisms was performed as

described by Kaech & Egli (2001).

Carbon balances using 14C-Iabelled TM. Carbon balances for P. putida TM 1 cultures were

performed as follows. During the exponential phase of a culture growing with TM (conditions

and medium as described by Kaech & Egli, 2001), 15 ml were transferred into a 50 ml

Erlenmeyer flask and incubated at room temperature. Aeration of the culture was achieved

using a magnetic stirrer. After the addition of 14C-labelled TM, the flask was sealed with a

silicon stopper. The culture was bubbled with air and the air outlet was connected to two

vessels (7 ml) in sequence containing each 5 ml of Carbo Sorb (Packard Bioscience

Company, Groningen, The Netherlands) to absorbe the produced CO2. Samples of the culture

and the C02-absorbing liquids were taken immediately after addition of the 14C-Iabelled TM

and after incubation for three hours. 14C-Iabel incorporated into biomass was determined by

filtering 2 ml of the culture liquid through a 0.45 /1m cellulose nitrate membrane filter

91

Chapter 5

(Millipore, Volketswil, Switzerland) using a vaccum pump. Filters and collected cells were

washed with distilled water containing 15 mM cold TM and thereafter dissolved in 3 ml of

Filtercount scintillation cocktail (Packard Bioscience Company). Samples of the filtrates and

the culture broth were transferred to scintillation vials containing 4 ml of Instagel plus

(Packard Bioscience Company) and aliquots of the CO2-absorbing vessels were put into

scintillation vials containing 5 ml of Permafluor E+ scintillation cocktail (Packard Bioscience

Company). The radioactivity obtained in the different fractions was then determined in a

liquid scintillation analyser (Tri-Carb 2200 CA, Packard, USA).

Preparation of cell-free extracts. The preparation of the cell-free extracts of P. putida TM 1

and the isolate DM 2 were performed as described in Chapter 4.

Enzyme activity assays. Enzyme activity assays were carried out in GC-Vials (Supelco Inc.,

Bellefonte PA, USA) of 5 to 20 ml volume. Protein solutions were stirred with a magnetic

stirrer and incubated at a pH of 7 in a 30 QC water bath. Using NMR spectroscopy as the

analytical method, the samples were either diluted with D20 (Aldrich Chemical Company

Inc., Milwaukee Wi, USA) or PB in D20 was used in the assay. The concentration of protein

used in assays was always in the range of 0.4 to 1.7 mg mrl. To stop the reaction, samples

removed from the assay were either heated for 1 min in a water bath of 95 QC or HCI 1 M was

added (l0 % of sample volume). Before analysis, acidified samples were neutralised with

NaOH 1 M (10 % of sample volume) and the pH was controlled with test strips (Tritest

pH 1 - 11, Macherey-Nagel, Dtiren, Germany). The stop procedure performed with acid did

not change the chemical composition of the samples as observed by lH NMR analysis. To

exclude oxygen, experiments were carried out in sealed vessels with a continuous nitrogen

flow through the headspace. All chemicals used in these experiments, except for the protein

stock solution, were bubbled with nitrogen for 10 min before adding them to the assay. NAD+

and phenazine methosulfate (PMS) were tested as electron acceptors. They were prepared as

stock solutions and the final concentration used in individual assays is indicated at the

appropriate place. For control experiments, the protein solution was heated in a water bath

(-95 QC) for 1 min prior to the addition of the different substrates. Samples removed from

assays were stored at -20 QC prior to analysis. Enzyme assays were also carried out directly in

92


NMR-tubes (diameter: 5 mm) in the NMR spectrometer and IH NMR spectra were acquired

as a function of reaction time. Between the acquisition of lH NMR spectra, the tube was

rotated to mix the protein solution, since the PF sank to the bottom of the tube without

agitation.

Assays to check the presence of TMA dehydrogenase or monooxygenase in CFE and SF were

performed as described by Colby & Zatman (1972). Samples were analysed by UV-VIS

spectrophotometry and by IH NMR spectroscopy.

To check the presence of formaldehyde dehydrogenase, the following enzyme assays were

performed. To 1 ml of 100 mM sodium phosphate buffer (pH 8) in a quartz cuvette, NAD+

and glutathion were added to a final concentration of 2 mM and 3 mM, respectively.

Subsequently, the protein fraction to be tested (0.14 mg mrl final concentration in the assay)

and formaldehyde (1 mM final concentration) were added, and the absorption of NADH was

monitored at 340 nm in a spectrophotometer. Formate dehydrogenase was measured by a

similar procedure. However, the pH of the phosphate buffer was 7.5, glutathion addition was

omitted and sodium formate (pH =7) was used as substrate (1.7 mM final concentration). To

calculate specific activity the initial slopes and an extinction coefficient of 6200 M'I cm'l for

NADH was used. All assays were done at 30°C.

Specific activities are given in nmol of converted substrate per min and per mg of protein

(nmol min'l mg'I).

To determine protein concentration the Bio-Rad enzyme assay was used according to the

manual of the manufacturer (Bio-Rad Laboratories GmbH, Munich, Germany). BSA was

used as standard (A-7906, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

Filtration. Cell constituents and proteins were removed from samples by filtration. For small

volumes (0.5 ml), the samples were centrifuged at 13000*g in a microcentrifuge at room

temperature using Ultrafree centrifugal filter devices (Biomax, cut-off 5000 Da, Millipore

Corporation, Massachusetts, USA). For volumes of 10 ml, larger Centriplus centrifugal filter

devices (YM-3, cut-off 3000 Da, Millipore) were used, centrifuging at 3000*g at room

temperature for several hours. Since the filter membranes were humidified with glycerol, all

filter units were prewashed several times with distilled water. As controls samples of the last

93

Chapter 5

washing step were analysed with the same method as the samples obtained from the enzyme

assays.

Thin layer chromatography (TLC). Samples from enzyme assays performed with 14C_

labelled TM as a substrate were separated by thin layer chromatography (TLC) using silica

gel coated aluminium plates (DC-Alufolien Kieselgel 60 F254, 20 x 20, Merck KgaA,

Darmstadt, Germany). Methanol: ethylacetate (1:1) or acetone: NaCI 1 M (1:1) were used as

eluents. Appropriate amounts (5 - 20 J.tl) of the samples were either directly applied onto the

TLC plates or proteins were removed previously by filtration. After chromatographing the

plates in a closed and eluent saturated TLC chamber (incubation time about 1 hour), they

were dried at room temperature and exposed to a Biomax film (Eastmann Kodak Company,

Rochester, New York, USA) for 4 to 7 days at -80 QC using a cassette with an intensifying

screen. Afterwards, films were developed in an automatic Kodak developer.

Extractions. Metabolites from enzyme assay samples were extracted with diethylether,

methyl acetate, and ethyl acetate. The volume of the solvent used was twice that of the

sample. When two extraction steps were performed the organic phases were combined for

analysis. From extractions carried out with 14C-Iabelled TM (propyl- or methyl-labelled) the

water phase (before and after extraction), the interphase and the organic phase were analysed

by scintillation counting. The interphase was analysed as well because a considerable amount

of TM aggregated in this layer. Aliquots of the samples and extracts were added to 3 ml of

Insta-Gel Plus scintillation cocktail (Packard BioScience Company, Groningen, The

Netherlands) and the radioactivity was measured in a liquid scintillation analyser (Tri-Carb

2200 CA, Packard, USA). Based on this, the extraction efficiency was calculated. Control

experiments were performed with inactivated protein solutions.

Desalting. Desalting was performed to remove the sodium phosphate buffer and other

charged components from the sample. A desalting chamber of 2.5 ml volume with a cation

and an anion-selective membrane (Berghof, Enningen, Germany) was used. A cooled (4 QC),

circulating NaCI solution (0.5 g r1) served as electrolyte. Power (final voltage: 500 V) was

applied to the chamber until the power supply indicated no current anymore.

94


Lyophilisation. Freeze-drying was applied to concentrate product components from the

different fractions (original samples, protein-free samples and/or desalted samples). The

solutions were frozen at -80 QC and subsequently dried in a Lyovac GT2 (Leybold AG, Koln,

Germany).

Nuclear magnetic resonance spectroscopy (NMR). IH NMR spectroscopy was used as the

analytical method to follow enzymatic activity and to determine the reaction products.

IH NMR spectroscopy was performed as described in Chapter 4. The chemical shifts of TM

and choline relative to 3-trimethylsilyl-tetradeutero-propionate are listed in Table 4.1,

Chapter 4.

Ion-pair chromatography. Additionally to IH NMR spectroscopy, TM was measured by

ion-pair chromatography as described by Kaech & Egli (2001).

Spectrophotometric determination of TMA. TMA concentrations in batch cultures were

determined by the spectrophotometric method as described by Shen (1988).

GC-MS (gas chromatography coupled with mass spectrometry). For the analysis of

expected products GC-MS was performed using a gas-chromatograph Fisons 8000 with PTV

injector OPTIC 2 (ATAS, Veldhoven, The Netherlands) and a high resolution mass

spectrometer (Autospec S, Micromass, Manchester, UK). GC-parameters: 1 J.tl injection

volume; column: XTI-5, 30 m, 0.25 mm internal diameter, 0.25 J.tm film thickness (Restek

Corp., Bellefonte, PA, USA) with a 2 m pre-column Siltek of 0.53 mm internal diameter

(Restek); oven temperature program: 50 QC for 2 min, gradient 9 QC min'I to 320 QC, followed

by 320 QC for 5 min, giving a total run time of 37 min. Injector parameters: Equilibration

time, 0.00 min; initial temperature, 270 QC; ramp rate, 0 QC min,I; final temperature, 270 QC;

splitless time, 1.5 min; transfer pressure, 0.85 bar; transfer time, 1.5 min; initial run pressure,

0.85 bar; final run pressure, 2.0 bar; end time, 55 min; purge gas flow, 2 ml min-I; split gas

flow, 50 ml min'I. MS-parameter: Ionisation-mode, +EI; acceleration voltage, 8000 V;

transferlines, 300 QC; source temperature, 280 QC; electron energy, 70 eV; trap current,

95

Chapter 5

500/lA; detector, 270 V; calibration, perfluorokerosene low boiling; resolution, 1000 (5 %

Valley); mass-range, 60-500 Da; scan time, 0.9 s; delay time, 0.5 s.

The ethylacetate phase of extracted samples was directly injected into the system. Freeze

dried samples of enzyme assays were dissolved in a mixture of ethylacetate (200 ILl) and

methanol (300 ILl) before injection. For derivatisation of formed metabolites a mixture of

N-methyl-N-trimethylsilyltrifluoroacetamid, l,4-dithioerythritol (Merck, Darmstadt,

Germany) and l-(trimethylsilyl)imidazole v:w:v = 1000:2:2 was used. To perform the

reaction 50 ILl of the reagent was added to the freeze-dried samples and incubated 30 minutes

at 60 cc. Afterwards, the derivatisation reagent was evaporated with nitrogen and 200 ILl of

hexane was added to dissolve the products that might have been formed.

ESI-MS (Electrospray ionisation mass spectrometry). Samples of enzyme assays were also

analysed by direct infusion ESI-MS (TSQ Quantum, Finnigan, CA, USA). ESI parameters

were: Infusion rate, 40 ILl min- I; spray voltage, 4000 V (ESI+), 2500 V (ESr); sheath gas

pressure, 39 psi; auxiliary gas pressure, 5 psi; capillary temperature, 350 cC; tube lens offset,

61 V. Scan parameters: Mass range, 50 - 500 Da; scan time, 0.75 s; peak width, 1.00 Da.

RESULTS

Conversion of TM. Disappearance of TM was observed in cell-free extract and in the

particulate fraction of P. putida TM 1 cells grown with TM as sole source of carbon, energy

and nitrogen. In contrast, TM was neither consumed in the soluble fraction independent of the

presence or absence of 2 mM NAD+, nor in control experiments with heat-inactivated

extracts, indicating that TM-transforming enzymes in P. putida TM 1 are membrane

associated. Addition of cofactors or cosubstrates was not required for TM-consuming activity

in the PP. Even under anoxic conditions TM was converted. This points to a lyase

mechanism. The addition of PMS (2 mM) did not show an effect on the reaction and NAD+

(2 mM) even inhibited consumption of TM. In enzyme assays with initial TM concentrations

of 50 mg r l up to 6 g r l the reaction always levelled of when 29 ± 5 % (6 values) of TM had

been consumed, independent on the protein concentration used (between 0.4 and 1.7 mg mr l).

The extent of - 30 % conversion was not caused by enantioselectivity of the enzyme system,

96


since a 1 : 1 racemic mixture of TM was used and therefore would yield 50 % of TM being

consumed. When initial concentrations of 10 and 15 g r 1 of TM were used, the amount of

consumed TM was reduced to 15 % and 10 %, respectively, even after extended incubation

times of up to 24 hours. With initial TM concentrations of 50 to 600 mg r1 the final level of

TM consumption was reached after approximately 1.5 hours (Figure 5.1). The initial specific

activity in enzyme assays with 1.5 mM of TM was 4 - 5 nmol min-1 mg-1 only, based on IH

NMR spectroscopy (Figure 5.1) and ion-pair chromatography data.

0"..... 0

o 08 0 0

/ t ~ ~

--

2.0

1.5

~.s« 1.0:2I-

~I-

0.5

0.0o 2

Time [h)3 4

Figure 5.1. Disappearance of TM (0) and appearance of TMA ( ... ) in the particulate fraction of

P. putida TM 1 grown with TM as sole source of carbon, energy and nitrogen, as determined by

IH NMR spectroscopy. Values are based on the absolute integrals of the CH3-signals in the IH NMR

spectra. Initial TM concentration: 1.5 mM. Protein concentration used in the assay: 1.7 mg ml-1• The

specific activity based on the initial slope was 4.4 nmol min-1 mg-1 (solid line). Note: The final TM

level was reached already after 80 min of incubation with only 20 % of TM being converted. Since the

experiment was carried out directly in an NMR tube in the NMR spectrometer, the mixing most

probably was insufficient (only rotation of the tube between data acquisition procedures) and,

therefore, the extent of TM conversion of roughly 30 % typically observed in experiments with intense

stirring was not reached.

97

Chapter 5

Trimethylamine. In IH NMR spectra recorded during enzyme assays with PF and CFE,

TMA was detected as a product of the consumption of TM and TMA was formed

stoechiometrically to the disappearance of TM (Figure 5.1). Since TMA possesses nine

equivalent protons (as does TM), the IH NMR spectra provided a clear an intensive singlet

(Figure 5.2). Because only approximately 30 % of the originally present TM was converted,

TMA and the suspected product of the reaction (i. e. glycidol, see section "Unidentified

products") were investigated with respect to their influence on the extent of conversion. Either

TMA (1.5 mM) or glycidol (1.5 mM) were added to the assay before starting the reaction with

TM (1.5 mM). Astonishingly, no product inhibition or change in the percentage of consumed

TM was detected. Also readdition of the same amount of PF to an assay with 1.5 mM TM

after the reaction had levelled off lead to no further degradation of the residual TM.

(a)/ OH

" ~N~OH""-/ TM

I3.30

I3.20

I3.10

I3.00

I I2.90 8(1H) ppm

Figure 5.2. IH NMR spectra ofthe enzyme assay shown in Figure 5.1 after (a) 2 min and (b) 80 min

of incubation. Initial TM concentration: 1.5 mM. Protein concentration of the particulate fraction of

P. putida TM 1 grown with TM as sole source of carbon, energy and nitrogen: 1.7 mg ml-1. Only the

section of the CH3-group signals is depicted. Arrows show the assignment of the signals to the

corresponding group in TM and TMA, respectively.

98


Based on these observations above, several experiments were carried out to examine whether

or not in the PF of P. putida TM 1 formation of TMA from TM was an artifact or a

physiological property. The degradation of TM was investigated in cell-free extracts of

P. putida TM 1 grown with either acetate or choline but neither in the CFE nor in the PF of

these cells TMA was formed in enzyme assays using 15 mM of TM. This suggests that the

enzymes acting on TM are inducible and that the reaction is not an artifact of the PF itself. PF

also was prepared from isolate DM 2, belonging to a different bacterial genus, when grown

with TM as sole source of carbon, energy and nitrogen (Chapter 3). In an enzyme assay using

this PF (l mg mr l of protein) and a TM concentration of 1.5 mM of TM, TMA was also

formed and NAD+ inhibited the reaction as well.

TMA formation was also investigated with growing cells of P. putida TM 1. Additional TM

(30 mM) was either pulsed to a culture grown with 3.7 mM of TM in the stationary phase, or

cells from the exponential growth phase of a batch culture growing with 30 mM of TM were

harvested by centrifugation and suspended in new medium containing 30 mM of TM. In both

experiments the time course of TMA concentration in the medium as well as the optical

density were monitored. The results obtained when resuspending the cells in fresh medium

are shown in Figure 5.3.

1.0 0.35

0.9 0.30

0.250.8

E 0.20 ~c: .sco<t 0.7l!l «Cl

0 0.15 ~I-

0.60.10

0.5 0.05

0.4 0.00

0 2 3 4 5 6 7 8 9Time [h]

Figure 5.3. Time course of the optical density ( 0 ) and the TMA concentration ( ... ) in a batch culture

of P. putida TM 1 after resuspending cells harvested from the exponential phase of a batch culture in

fresh medium. The initial TM concentration was 30 mM in both cultures.

99

Chapter 5

In both experiments excretion of TMA into the culture medium was detected, confirming that

TMA formation from TM did also occur in living cells of P. putida TM 1. Although, the

maximum concentration of TMA excreted into the medium was very low, ~ 0.1 mM in pulse

experiments, and ~ 0.3 mM after resuspension (Figure 5.3), the formation of TMA was

unambiguous. Undisturbed batch cultures, even with NH4CI (800 mg r1) as additional

nitrogen source, and chemostat cultures of P. putida TM 1 growing with TM never contained

detectable levels of TMA.

If TMA was a metabolite in TM degradation, one would expect that TMA can be used as a

nitrogen source by the organism. This was tested in batch and continuous cultures of P. putida

TM 1 with either acetate or ethanol as additional carbon source and TMA as the sole source of

nitrogen (both, acetate and ethanol can be used as carbon sources for growth by

P. putida TM 1; Kaech & Egli, 2001). For this, 10 % (v/v) of a P. putida TM 1 batch culture

(3.7 mM TM) from the exponential phase was transferred into a shake flask containing fresh

medium with 17 mM of acetate and 3 mM of TMA at a pH of 9. A batch with 17 mM of

acetate only was used as a control. A pH of 9 was chosen to increase the concentration of non

protonated TMA available (~ 14 % of total TMA is present in the neutral form at pH = 9) that

is expected to enter the cells by simple diffusion. No difference was found between the batch

with TMA as nitrogen source and the control, strongly suggesting that TMA was not used as

nitrogen source. A slight growth was observed in both cases and may be caused by the

residual nitrogen transferred with the inoculum (cells not washed) and/or by an accumulation

of surplus carbon as storage material.

The utilisation of TMA as a nitrogen source also was investigated by pulsing TMA to a

nitrogen-limited continuous culture of P. putida TM 1 at a dilution rate of 0.1 h-1 and fed with

2.6 mM (350 mg r 1) TM and 17 mM (l g r 1

) ethanol at 30 QC and a pH of 8 (~2 % of total

TMA present in the non-protonated form). TM was provided as a substrate to guarantee the

expression of all enzymes required for TM utilisation. Ethanol as an additional carbon source

was used to ensure the assimilation of surplus nitrogen from TM, usually released as

ammonium. The ratio of TM : ethanol was set such that neither residual TM nor excreted

NH/, but residual carbon (~ 850 mg r1) was present. When the steady-state was reached,

TMA was pulsed to the culture to a final concentration of 0.85 mM and the biomass (optical

density), dissolved organic carbon and ammonium were measured before the pulse and during

100


8 hours following the pulse. The ammonium concentration remained below the detection

limit, additional excess ethanol was not utilised (this would have resulted in a decrease in

DOC) and the biomass concentration remained constant. This clearly demonstrates that also

under these conditions TMA was not utilised as a nitrogen source. As a control,

monomethylamine (MMA) was pulsed to the same culture (final concentration 1.4 mM),

since MMA was known to be used by P. putida TM 1 as a nitrogen source in batch cultures.

Four hours after the pulse, the biomass started to increase dramatically, confirming the

utilisation of MMA as a nitrogen source and the validity of the experimental setup.

Since TMA was formed in the PF, TMA-degrading activity was expected to be present in

CFE or SF of P. putida TM 1. Therefore, the assays for TMA dehydrogenase and TMA

monooxygenase originally developed for methylotrophs (Colby & Zatman, 1972) were used

to detect activity of TMA-utilising enzymes. However, no activity of neither enzyme was

found in CFE or SF ofTM-grown P. putida TM 1.

Unidentified products. Since TMA was removed enzymatically from TM in the PF of

P. putida TM 1 the product of this reaction has to be free of nitrogen. Based on chemical

reasoning, the missing part could be the epoxide glycidol and/or its hydrolysis product

glycerol because in the reaction neither oxygen nor other cofactors were required. However,

no indications for these compounds were found by IH NMR spectroscopy. By following the

degradation with high initial concentrations of TM, several signals in the IH NMR evolved

proportionally to TMA (Figure 5.4a and b). However, we were not able to attribute the

chemical shifts and integrals observed to any compound or product. The same signals were

found in IH NMR spectra of assays which were purified by filtration (removal of proteins)

and by desalting (removal of TM, TMA and buffer) and concentrated by freeze-drying

(Figure 5.4c). In IH NMR spectra of control assays done with inactivated protein solutions

these signals were absent. Therefore, these signals were attributed to the products formed,

which are obviously neither volatile nor charged.

101

Chapter 5

TM__--A---_

H20 ( --=~

(a):;.-- :;.-"-

TMAp

\ p

p

1p

I .1 ~ ~ 11 I

(b)TMA

p

p

p

p

(c)p p

PG

~

I I I I I I I I8CH) 5.0 4.5 4.0 3.5 3.0 2.5 2.0

I1.5 ppm

Figure 5.4. lH NMR spectra of untreated and treated samples deriving from enzyme assays performed

with the particulate fraction of TM-grown cells of P. putida TM I with TM as substrate. (a) lH NMR

spectrum of an untreated sample taken after 5 hours of incubation from an assay with 0.6 mg ml-1 of

protein and an initial TM-concentration of 45 mM. The assay was carried out using DzO-containing

buffer. (b) lH NMR spectrum of an untreated sample taken after 24 hours from the same experiment as

in (a). (c) lH NMR spectrum of a filtered, desalted and freeze-dried sample (dissolved in DzO) taken

after 2 hours from an assay with 0.85 mg ml-1 of protein and an initial TM-concentration of 7.5 mM.

P: Signals of potential products. G: Residual glycerol from membrane filter used for the removal of

the PP.

102


To obtain more information on the properties of the product(s), enzyme assays were

performed with radioactive TM, using 14C-propyl-Iabelled TM. Aliquots taken after 2 hours

of incubation from assays with PF and from control assays with inactivated PF were run on

silicagel thin layer chromatography plates using either acetate: NaCI (1 M) or

methanol: ethyl acetate, both 1:1 (v/v), as eluents. The plates were then incubated on

radiosensitive films (Figure 5.5).

In this way the presence and formation of at least two products deriving from the propyl

group of TM was demonstrated. Samples were also freeze-dried and subsequently dissolved

in methanol before carrying out TLC. The same picture as shown in Figure 5.5 resulted,

confirming again that these products were not volatile.

Samples of the assays performed with 14C-Iabelled compounds were also extracted with

different solvents, such as ethyl acetate, diethyl ether and methyl acetate. Significant amounts

(a)

Acetone: NaCI (1 M)

1 : 1

(b)

Flow

Start

Ethylacetate : MeOH

1 : 1

Figure 5.5. Separation of educts and products from enzyme assays using the PF of P. putida TM 1 by

thin layer chromatography (silicagel coated alu-plates). (a) Acetone: NaCl (1 M) and (b) ethylacetate :

methanol, both 1:1 (v/v), were used as eluents. Samples were taken from active PF and heat

inactivated PF control assays with a protein concentration of 1.2 mg rnI-1and about 0.15 mM of 14C_

propyl-labelled TM. Lane 1 and 2: Samples of the control (1) and active (2) assay taken immediately

after the addition of 14C_propyl TM. Lane 3 - 6: Samples of the control (3, 5) and active (4, 6) assay

taken after two hours of incubation.

103

Chapter 5

of the 14C-propyl radioactivity attributed to the unidentified products were extractable with

the solvent ethyl acetate only. In control assays with inactivated PF and in enzyme assays

with 14C-methyl-Iabelled TM, less than 1 % of the radioactivity was extractable with this

solvent. This demonstrates that neither the educt nor TMA was extracted with ethyl acetate.

Overall, in assays using 14C-propyl-Iabelled TM about 11 % of the label were found in the

ethyl acetate layer and was attributed to (an) unidentified product(s). With the other solvents

used either a very low percentage of the extracted products was detected in the solvent layer

(diethyl ether), or primarily the educt and TMA were extracted by the solvent (methyl ether).

Purified and concentrated samples (filtered, desalted and freeze-dried) were analysed by

GC-MS, before and after trimethylsilyl derivatisation. In addition, the ethyl acetate phase of

the extraction procedure was injected in the GC-MS (underivatised). However, no significant

signals of a potential product were detected in any of the samples. Direct infusion into a mass

spectrometer (ESI-MS) of untreated samples was also performed, but this only provided the

educt signal. Therefore, with the exception of TMA the product(s) formed from TM by the

membrane-associated TM-Iyase remain unidentified so far.

Substrate specificity. The substrate specificity of the TM-transforming enzyme system in the

PF of P. putida TM 1 was tested for its activity with several structurally similar compounds.

These included the QAAs DM and MM, which are also used as head groups in esterquat

surfactants, as well as the related compounds L-, D-camitine, betaine and choline. All enzyme

assays were performed under the same conditions as those used for TM, i. e. with initial

substrate concentrations of 1.5 mM under aerobic conditions and without the addition of any

cofactors or cosubstrates. Judged from I H NMR spectroscopy analysis only choline was

converted in the PF of P. putida TM 1, but TMA was obviously not produced. Evaluation of

the I H NMR spectra provided two oxidation products of choline, namely betainealdehyde and

betaine (Figure 5.6). These structures were confirmed using pure betainealdehyde and betaine

standards (for details on the analysis, see chapter 4). In the PF of P. putida TM 1 cells grown

with choline as sole source of carbon, energy and nitrogen, choline was converted in the same

way. However, no TM-transforming activity was detected in such extracts.

104

Betaineo

lJ-°H/\

Betainealdehyde(hydrated form)

OHIJ-OH

/N\

\


8(1H) 3.28 3.26 3.24 3.22 3.20 ppm

Figure 5.6. IH NMR spectrum with assignments of the methyl groups of choline and its degradation

products betaine and betainealdehyde. The sample for IH NMR spectroscopy was taken after 2 hours

of incubation of the PF (1.1 mg ml'l of protein) ofTM-grown P. putida TM 1 with 1.5 mM of choline.

Only the section of the CH3-group signals is shown.

Fate of the TM carbon in growing cells of P. putida TM 1. Carbon balances in batch

cultures of P. putida TM 1 with l4C-labelled TM were performed to investigate the fate of the

carbon atoms in TM. When l4C-methyl-labelled TM was used, - 25 % of the utilised TM

carbon was incorporated into biomass and - 75 % was combusted to C02. With l4C_propyl_

labelled TM, even - 92 % of the converted TM ended up in CO2 and only - 8 % were

incorporated into biomas (note: the propyl carbon in position 3 was labelled). Based on these

results and since P. putida TM 1 was not able to grow with Cl-compounds (Kaech & Egli,

2001), formaldehyde and formate utilising dehydrogenases were expected to be present in

cell-free extracts, mediating the oxidation of the methyl carbon to C02 and providing energy.

Therefore, the cell-free extracts of P. putida TM 1 were tested for activity of these widely

occurring enzymes. Indeed, high activity was found in the soluble fraction for both, NAD+

dependent formaldehyde and NAD+-dependent formate dehydrogenase, amounting to 362 ±

13 nmol min'I mg'I (7 measurements) and 130 ± 15 nmol min·I mg· l (5 measurements),

respectively. In control experiments with the CFE of acetate-grown cells, no formate

dehydrogenase activity, and only a low activity of formaldehyde dehydrogenase (30 nmol

min·I mg·I) was detected.

105

Chapter 5

DISCUSSION

All detected enzyme activities were associated with the membrane of strain TM 1 making

purification of the enzymes difficult or even impossible. Therefore, enzyme assays for the

investigations of the catabolic pathways were performed with the particulate fraction of strain

TM 1.

The initial degradation of TM is mediated by an inducible, membrane-associated lyase,

splitting TMA from TM (Figure 5.7a). This reaction was shown to be a physiological property

of P. putida TM 1 and no "in vitro" artifact even though externally provided TMA,

unexpectedly, was not used by growing P. putida TM 1 as a nitrogen source. The extent of

only 30 % of TM-conversion in enzyme assays using the particulate fraction was neither

caused by enantioselectivity of the enzyme system nor by an inhibition of the products. An

inactivation of the enzyme by a reactive (unidentified) product can also be excluded based on

the following "worst case" calculation. If the PF consisted solely of TM-active protein

(2 mg mr1) with a molecular weight of 10'000 Da (small protein) and each product molecule

inactivated one enzyme molecule, a consumption of 27 mg r 1 of TM might be reached only,

instead of a consumption of up to 1.8 g r 1 of TM as was observed.

In the literature the fission of the N-Calkyl bond was proposed by Van Ginkel (1996) as a

general strategy of microorganisms to degrade alkyl-amines and alkyl-trimethyl-ammonium

compounds. This mechanism was observed in Pseudomonads for hexadecyl-trimethyl

ammonium (Van Ginkel et aI., 1992), didecyl-dimethyl-ammonium (Nishihara et aI., 2000),

as well as for dodecyl-dimethyl-amine (Kroon et aI., 1994). However, the fission of the N

Calkyl bond was always catalysed by an oxidation reaction and the degradation of these

compounds was either restricted to a consortium of at least two microorganisms or was

incomplete resulting in the excretion of the corresponding amine into the culture medium. The

ability to split TMA from choline, different betaines and/or carnitines has also been reported

for various microorganisms and, where investigated, the fission was found to be mediated by

an oxidation reaction (Englard et aI., 1983; Kleber, 1997; Rebouche & Seim, 1998; Seim et

aI., 1982a; Seim et aI., 1982b; Unemoto et aI., 1966; Wood & Keeping, 1944). In contrast to

these reports, none of these quaternary trimethyl-ammonium compounds (i. e. choline, betaine

or L, D-camitine) was split to TMA and corresponding products in the PF of P. putida TM 1.

The N-Cethanol bonds of DM and MM were not split by the PF of TM-grown P. putida TM 1

106


either. Therefore, the lyase activity in P. putida TM 1 must be very specific and the presence

of a quaternary trimethyl-ammonium center in substrates is obviously not sufficient enough to

allow fission of the N-Calkyl bond.

(a)OH

lpOH I[ ? ]

I ~OH/\ •

/N"",+

oGlycidolTM Trimethylamine

: .,.H2OI "

? l0 / [HO~OH]

H)lHHN

'" Gly::rolFormaldehyde Dimethylamine

: .,.ATPI "

~n ? l2 e-+ 2 H+

0 t 0 JH)lOHH

2N- \\/OH

PHO~O/\

Formate MethylamineG~~erol-3P OH

~mI

? III

2 e-+ 2 H+ •CO2 NH3

Central metabolism

(b)

2 e-+ 2 H+ HO

l0°~. IJ-OH/ \ -/~ /N\

Choline H20 Betainealdehyde(hydrated form)

Betaine

Figure 5.7. Ca) Proposed degradation pathway of TM: I) "TM-Iyase", II) NAD+-dependent

formaldehyde dehydrogenase, Ill) NAD+-dependent formate dehydrogenase. Cb) Oxidation of choline

in the particulate fraction of TM-grown P. putida TM 1.

107

Chapter 5

If the first step in TM catabolism in cells of P. putida TM 1 leads to the production of TMA,

and this metabolite is not excreted into the medium but used as a source of nitrogen by the

bacterium, one must expect TMA to be demethylated in some ways (Figure 5.7a). However,

neither TMA dehydrogenase nor monooxygenase activity was found employing the classic

assays described in the literature for methylotrophs (Colby & Zatman, 1972). The inability to

detect activity does not exclude the involvement in particular of TMA monooxygenase in

P. putida TM 1 because these enzymes are known to be very labile (Boulton et aI., 1974). The

removal of methyl groups from TMA is indicated by the high activity of inducible, NAD+

dependent formaldehyde and formate dehydrogenase in the soluble protein fraction,

suggesting that the methyl groups were oxidised to CO2 rather than incorporated into biomass

(Figure 5.7a). This suggestion is supported by the observation that P. putida TM 1 was unable

to grow with Cl-compounds (Kaech & Egli, 2001). Carbon balances with 14C-methyl-Iabelled

TM confirmed the complete oxidation of the methyl groups, since 75 % of the methyl carbon

was transformed to C02. Astonishingly, not only the Cl groups but also the 14C_propyl_

labelled carbon was almost exclusively combusted to CO2, this carbon atom of the molecule

even to an extent of 92 %. These results correspond with the low growth yield recorded in

batch cultures of P. putida TM 1 and the high percentage of 60 % of the total carbon present

in this compound combusted to CO2 (Kaech & Egli, 2001).

Considerable effort was put into the identification of the remaining part of the TM molecule.

The formation of at least two products, neither charged nor volatile, was demonstrated but

elucidation of their structures by performing GC-MS, ESI-MS and IH NMR was not

successful. Future investigations should focus on separating and concentrating these

compounds. Best chances for success will probably involve the development of an appropriate

liquid chromatography method, coupled to mass spectrometry.

With respect to the substrate specificity of the TM-transforming enzyme system in the PF of

P. putida TM 1 grown with TM, only choline was converted, whereas DM, MM, betaine and

carnitine remained untouched. However, when compared to TM, the conversion of choline

was achieved by a totally different mechanism. Choline was oxidised to betainealdehyde and

betaine in the PF of P. putida TM 1 indicating the presence of a membrane-associated choline

oxidoreductase mediating both oxidation steps (Figure 5.7b). The same activity was found in

the PF of isolate MM 1, too (Chapter 4). The oxidation of choline to betainealdehyde and

108


betaine was reported in the literature to be the most frequently occurring pathway for choline

degradation in bacteria (Kortstee, 1970; Shieh, 1964). Several membrane-associated, choline

specific enzymes are documented in the literature for Pseudomonas aeruginosa and

Escherichia choli (Bater & Venables, 1977; Lamark et aI., 1991; Nagasawa et al., 1976;

Russell & Scopes, 1994). Interestingly, all these choline oxidoreductases (dehydrogenases)

only oxidised choline to betainealdehyde, but did not catalyse a second oxidation step to the

acid (betaine) as found in this study. Since two completely different mechanisms in the PF

were responsible for the initial degradation of choline and TM and because TM was not

converted in the PF of choline-grown cells, the enzymes acting on choline and TM are

obviously distinctly different.

ACKNOWLEDGEMENTS

We thank Hans-Ruedi Aemi and Rene Schonenberger for the GC-MS and ESI-MS analysis.

109

ite Leer /Blank leaf

Concluding remarks

6. Concluding remarks

Detailed discussion of the results can be found in the individual chapters. Here, I would like

to point out and add some conclusions and hypotheses related to the questions addressed at

the beginning of this work.

Competent bacteria. With all three quaternary ammonium alcohols TM, DM and MM,

microorganisms were isolated that were able to grow with one or more of these QAAs as a

sole source of carbon, energy and nitrogen. So far, the degradation of quaternary ammonium

compounds was supposed to be achieved exclusively by consortia of at least two different

microorganisms (Van Ginkel, 1996). However, this hypothesis was based on investigations of

mainly long alkyl chain quaternary ammonium compounds whereas the QAAs investigated in

this study contained short hydroxylated alkyl substituents and the properties of these short

substituents may be the key to this issue. Anyhow, the general view that consortia are

required for complete degradation of quaternary ammonium compounds must be questioned.

In contrast to earlier reports the QAAs were provided here as the sole source of carbon,

energy and nitrogen and - probably because of this enrichment strategy - the isolated

microorganisms were able to completely catabolise the individual QAAs. Nevertheless, in

batch cultures not each of the isolated strains degraded all the QAA supplied to completion,

yielding exclusively biomass, NH/ and CO2. Strain MM 1, when cultivated with MM,

excreted considerable amounts of dead-end metabolites (Figure 6.2), and strain DM 1 used

only a part of the DM supplied (why is not known, tests done for limitiations in the growth

medium indicated excess of all essential nutrients) and, therefore, these compounds have the

potential to accumulate in the environment. These findings could have considerable impact on

the design of new similar compounds with respect to enhanced environmental properties, i. e.,

ready and ultimate biodegradation.

Interestingly, standard biodegradation tests with activated sludge or waste water treatment

effluent (Hales, 1998; Krueger et aI., 1998; Puchta et al., 1993; Waters et al., 1991) suggested

degradation to completion for all three QAAs, TM, DM and MM, forming biomass and CO2

of the carbon provided. Complete degradation in complex systems could be to due not only to

strains similar to these isolated here able to degrade QAAs intracellularly to completion, but

to simultaneous consumption of additional carbon sources to balance the nitrogen excess of

QAAs (see Egli, 1995), or additional microorganisms degrading metabolites from QAAs

excreted by "incomplete degraders". This raises the question, whether or not the microbes we

111

Chapter 6

have isolated and characterised are the same as those that do the job in the environment. To

answer this question, however, further investigations are required. Adequate methods to

investigate the abundance of the isolated bacterial strains in environmental systems exposed

to QAAs would be the use of 16S-rRNA probes for fluorescent in situ hybridisation, since

16S-rDNA was sequenced for all isolated strains, or surface antibodies like used for NTA

degrading bacteria (Bally et aI., 1994; Wilberg et aI., 1993).

The four isolated characterised strains differ considerably with respect to their physiological,

nutritional and biochemical properties. This supports and confirms the findings for the

degradation of the three QAAs in standardised biodegradation tests reported by Hales (1998),

who observed different degradation patterns. Phylogenetically, i. e. based on 16S-rDNA

similarity, the isolated bacteria belonged not only to different microbial genera but the isolates

are positioned in each of the major bacterial groups of protobacteria (Figure 6.1). The ability

to degrade TM, DM or MM is obviously not a trait of a single bacterial genus or even a

particular species.

Surprisingly, only one of the isolated strains, named DM 1, was able to grow with another

QAA (TM) than that used for its isolation, whereas all of them were able to degrade the

natural, structurally related compound choline. Vice versa, none of the tested established

reference choline degraders P. putida DSM 291T and Z ramigera Itzigsohn 1868AL or several

bacterial strains isolated in the course of this work with choline were able to degrade any of

the QAAs. Hence, the ability to degrade TM, DM or MM appears to be a specific property of

microorganisms and the competence to degrade choline does not necessarily imply the ability

to degrade one of the QAAs derived from esterquat surfactants. Therefore, the design of

QAAs with a structure similar to that of choline is not a priori a prerequisite for favorable

environmental properties.

112

Concluding remarks

Proteobacteria

a Subdivision

AcetobacteraceaeCau/obacter groupHyphomonas groupRhizobiaceae groupRhodobactergroup ------------------------------------ Isolate MM 1 (new genus?)Rhodospirillaceae MM-oxidoreductaseRickettsia/es (choline oxidoreductase?)SphingomonadaceaeUnclassified alpha proteobacteria

~ Subdivision

A/ca/igenaceae iAmmonia-oxidizing bacteria :--Burkho/deria/Oxa/obacter/Ra/stonia group El

Comamonadaceae Isolate OM 1 (new species?)Gallionella group , Isolate OM 2 (new species?)Hydrogenophilus group I I..-

Massilia El l--Methy/ophilus group J= Zoog/oea ramigeraNeisseriaceae Zoog/oea resiniphilaRhodocyc/us group --------'. Zoog/oea Zoog/oea sp.Spirillum groupUnclassified beta proteobacteria

y Subdivision

AeromonadaceaelSuccinivibrionaceae groupAlishewanella groupA/teromonadaceae groupCardiobacteriaceae groupChromatiaceaelEctothiorhodospiraceae groupCuracaobacter groupEnterobacteriaceae groupLegionellaceae groupMethy/ococcaceae group ~OceanospirillumlHa/omonas groupPasteurellaceae groupPseudomonaceaelMoraxellaceae group P. putida -- P. putida TM 1SUlfur-oxidizing symbionts TM-IyaseThiothrixlFrancisella groupVibrionaceae groupXanthomonada/esUnclassified gamma proteobacteria

o/E Subdivision

Unclassified proteobacteria

Figure 6.1. Phylogenetic tree of the proteobacteria according to the National Center for Biotechnology

Information (NCBI, www.ncbi.nlm.nih.gov)andpositionsoftheisolatesTM1.DM1.DM 2 and

MM 1.

113

Chapter 6

Initial degradation steps. Consumption of MM and TM was observed in the cell-free

extracts of the strains MM 1 and TM 1, respectively. Figure 6.2 gives an overview over the

initial degradation steps of MM, TM and choline in the microbial isolates based on the

observations in this work. Completely different enzymatic mechanisms were responsible for

the degradation of these two QAAs confirming again the different degradation patterns found

for the QAAs in standardised biodegradation tests (Hales, 1998).

The initial degradation of MM in strain MM 1 was mediated by a membrane-associated,

constitutively expressed oxidoreductase. Since also DM and choline were oxidised exhibiting

the same characteristics, the responsible enzyme for this reaction was probably the same

enzyme. Two observations suggest that this enzyme may be a choline oxidoreductase with

extended substrate specificity. First, its ability to degrade choline, second that it is

constitutively expressed during growth with choline, MM or acetate. Hence, the initial

degradation of MM is most probably linked to the degradation of choline. Investigation of this

relationship could be performed by producing choline-deficient mutants of strain MM 1 and

checking them for the ability to degrade MM. DNA sequences of several genes encoding

choline dehydrogenases and betainealdehyde dehydrogenases have been analysed (Lamark et

al., 1991; Pocard et al., 1997; Rosenstein et al., 1999), and could be a basis for investigations

at the genetic level.

The fact that TM was not transformed in the cell-free extracts of strain MM 1 can be

interpreted in two ways: Either, the presence of an ethanol group in the substrate is essential

to undergo oxidation in the enzyme and neither the presence of hydroxyl groups alone nor the

quaternary ammonium character of the compound is sufficient to evoke enzymatic oxidation

of hydroxyl groups; or the 2,3-dihydroxy-propyl part of TM, i. e. its steric dimension (Figure

6.2), prevents the compound to fit to the active site of the protein.

The initial degradation of TM in strain TM 1 followed a completely different mechanism.

Trimethylamine was split from TM by an inducible, membrane-associated lyase. None of the

structurally similar compounds DM, MM, choline, betaine or carnitine were accepted as

substrates by this enzyme system. However, choline underwent oxidation in the particulate

fraction of strain TM 1 providing betainealdehyde and betaine, as was also observed in strain

MM 1. Therefore, the trimethyl-ammonium structure is not sufficient to have a QAA accepted

as a substrate, suggesting that the lyase reaction is very specific. The same mechanism for the

initial TM-fission also was found in strain DM 2 isolated with DM, which was also growing

with TM. This strain belonged to a different genus, which leads to the speculation, whether or

not the degradation of TM is mediated predominantly or even generally by the mechanism

114

Concluding remarks

described above. However, to answer this question extensive screening of TM degrading

strains would be needed.

Interestingly, the enzyme(s) oxidising choline to betainealdehyde and subsequently to betaine

in both strains, MM 1 and TM 1, were membrane-associated. In contrast, either choline

oxidising enzymes found in microorganisms catalysing both oxidation steps were reported to

be soluble (Ikuta et aI., 1977; Ohta-Fukuyama et al., 1980) or a membrane-associated enzyme

was detected mediating exclusively the oxidation of choline to betainealdehyde (Bater &

Venables, 1977; Lamark et aI., 1991; Nagasawa et aI., 1976; Russell & Scopes, 1994).

In the cell-free extracts of the DM-growing strains, no transformation of DM was detected

and, hence, making investigations at the enzyme level impossible. The reasons may be due to

inactivation of the enzymes by the preparation procedure or the storage.

Obviously, for the initial degradation of TM and MM in bacteria, no general strategy (Van

Ginkel, 1995) can be proposed and no general relationship exists between (initial) QAA and

choline degradation. The mechanisms seem to depend on the specific, individual structure of

the quaternary ammonium alcohols TM, DM and MM. Although several indications were

found with respect to the environmental properties of the QAAs TM, DM and MM, no

general conclusions can yet be drawn concerning the design of new QAAs for cationic

detergents. Each new QAA requires extensive investigations of its biodegradability and of the

potential to generate dead-end metabolites that are excreted and perhaps recalcitrant.

115

Chapter 6

(a) (b)

TM

?

I [~OH]/N".... + 0

Trimethylamine Glycidol

?~OHN )l

'" H HFormaldehyde

}...NADHo

H)lOHFormate

}...NADHCO2

MM

HO~ +r-J0H

/N~OH

°HO~ OH

N+""""~/ v--- --. Excretion asdead-end metabolite

~2e+2H.

~.,.j~OH~/ v---0

~H20 Further

2 e- + 2 H+ breakdown

OH /

HO~ OH/

N+""""!\/ v---0

~2e-+2H.

(c)

~00H---L:+ IJ-OH/ \ . IIIr-~ /N\

Choline H20 Betainealdehyde(hydrated form)

2 e-+ 2 H+ HO.L IJo

~ /N\III /'

Betaine

Figure 6.2. (a) Initial degradation steps of MM in strain MM 1. I) Constitutively expressed,

membrane-associated MM-oxidoreductase. (b) Initial degradation step of TM in P. putida TM 1 and

proposed breakdown of trimethylamine. 11) Inducible, membrane-associated TM-Iyase. (c) Observed

oxidation of choline in strain MM 1 and P. putida TM 1. Ill) Membrane-associated oxidoreductase(s).

116

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